Defensins are effectors of the innate immune response with potent antibacterial activity. Their role in antiviral immunity, particularly for non-enveloped viruses, is poorly understood. We recently found that human alpha-defensins inhibit human adenovirus (HAdV) by preventing virus uncoating and release of the endosomalytic protein VI during cell entry. Consequently, AdV remains trapped in the endosomal/lysosomal pathway rather than trafficking to the nucleus. To gain insight into the mechanism of defensin-mediated neutralization, we analyzed the specificity of the AdV-defensin interaction. Sensitivity to alpha-defensin neutralization is a common feature of HAdV species A, B1, B2, C, and E, whereas species D and F are resistant. Thousands of defensin molecules bind with low micromolar affinity to a sensitive serotype, but only a low level of binding is observed to resistant serotypes. Neutralization is dependent upon a correctly folded defensin molecule, suggesting that specific molecular interactions occur with the virion. CryoEM structural studies and protein sequence analysis led to a hypothesis that neutralization determinants are located in a region spanning the fiber and penton base proteins. This model was supported by infectivity studies using virus chimeras comprised of capsid proteins from sensitive and resistant serotypes. These findings suggest a mechanism in which defensin binding to critical sites on the AdV capsid prevents vertex removal and thereby blocks subsequent steps in uncoating that are required for release of protein VI and endosomalysis during infection. In addition to informing the mechanism of defensin-mediated neutralization of a non-enveloped virus, these studies provide insight into the mechanism of AdV uncoating and suggest new strategies to disrupt this process and inhibit infection. Defensins are effectors of the innate immune response with antibacterial and antiviral activity. A major bactericidal mechanism of defensins is membrane disruption; however, their mechanism against non-enveloped viruses, such as human adenovirus, is poorly understood. This work shows that sensitivity of human adenovirus to defensins is species specific and that neutralization is dependent upon defensin tertiary structure. A cryoEM structural study of an adenovirus vector in complex with a neutralizing defensin, HD5, led to a neutralization model in which defensin binds to the interface of two capsid proteins, preventing dissociation of the fiber protein. We propose that binding at this site blocks downstream uncoating events required for infection. Infectivity studies using virus chimeras comprised of capsid proteins from sensitive and resistant human adenovirus serotypes support this model. This functional and structural study provides insight into the mechanism of human adenovirus neutralization by defensins and suggests new strategies to inhibit infection. Defensins are an evolutionarily conserved family of antimicrobial peptides that are an important effector component of the innate immune response. Humans express two classes of defensins, α- and β-defensins. There are six human α-defensins  and multiple β-defensins, which differ in their tissue distribution and expression patterns Defensins have also been shown to neutralize several non-enveloped viruses, including human adenovirus (HAdV), human papillomavirus (HPV), adeno-associated virus (AAV), and polyomavirus, despite the absence of a lipid target Our previous studies revealed the stage in the virus entry pathway that is blocked by defensins In our previous studies we showed that a small subset of HAdVs, including HAdV-5, -12, and -35  are neutralized by α-defensins D of this interaction is approximately 14.5 µM , which correlates reasonably well with the IC50 of HD5 for HAdV-5 infection (3–4 µM) Neutralization of HAdV by α-defensins is dependent upon binding to the virus capsid, which can be disrupted in the presence of elevated concentrations of sodium chloride We assessed HD5 binding to additional serotypes after incubation with 20 µM HD5 . The senTo gain further insight into the defensin-HAdV interaction, we assessed the requirement for two conserved structural elements on the anti-AdV activity of HD5. All defensins have three disulfide bonds . In addiPreviously we observed that HD5 enhances binding of HAdV-5 to cells despite an almost complete block to productive infection To obtain structural insight into the mechanism of α-defensin-mediated neutralization of HAdV infection, we studied a complex of Ad5.F35 (Ad35F) and HD5 by cryoelectron microscopy (cryoEM). This chimeric virus construct was chosen because of its short fiber and the availability of a cryoEM structure of Ad5.F35 in the absence of defensin for comparison In order to identify the binding regions for HD5 on the surface of Ad5.F35, we performed a difference map analysis using the available crystal structure of the HAdV-5 hexon (PDB 1P30) The difference map analysis reveals multiple binding sites for HD5 on the penton complex (penton base and fiber). The Ad5.F35 difference map clearly reveals the flexible RGD loop of penton base (78 residues) as well as the shaft and knob of fiber (yellow), which are missing from the penton base/fiber peptide co-crystal structure  and resistant  serotypes provided a means to test this HD5 neutralization model by generating virus chimeras. Initially, virus chimeras were constructed by replacing the sequences for fiber, penton base, and hexon in the HAdV-5 genome with the corresponding sequences from HAdV-19c. Consistent with previous studies Based on these results, we created an additional construct in which only the four residues in the HAdV-5 fiber variable region (DTET) were replaced by the corresponding residues from HAdV-19c (GYAR). Compared to HAdV-5, the GYAR virus is less sensitive to 5 µM HD5. We then combined the GYAR substitution with the PB substitution in a single construct (PB/GYAR). This construct, like 19cF and wild type HAdV-19c, is completely resistant to neutralization by HD5. This result confirms a role for the DTET/GYAR variable fiber region in HD5 neutralization, as the PB/GYAR chimera is even more resistant to HD5 then 19cPB alone. Additional studies using a higher concentration of HD5 (20 µM) confirmed the defensin-resistance of HAdV-19c, 19cF, and PB/GYAR (data not shown). Equivalent results were obtained using a FACS-based assay that requires 100-fold lower moi, indicating that variations in particle to pfu ratios among the virus preparations could not account for the differences in phenotype (data not shown). Studies equivalent to those in D that correlates well with the IC50 for virus infection, and antiviral activity is dependent upon the tertiary structure of a correctly folded α-defensin molecule. Structural analysis by cryoEM indicates that defensins bind to all of the exposed major capsid proteins. Based on sequence analysis and cryoEM studies, we proposed that potential critical sites for defensin binding are located at the point of contact between penton base and fiber. The importance of these sites for defensin neutralization was confirmed by an analysis of virus chimeras comprised of sequences from sensitive and resistant HAdV serotypes, indicating that neutralization determinants are found in both fiber and penton base. In conjunction with our previous studies, this observation suggests a model in which defensin binding to these critical neutralization sites prevents fiber dissociation, thereby blocking subsequent steps in HAdV uncoating that are required for release of protein VI, endosomalysis, and infection.These studies extend our understanding of the mechanism of α-defensin-mediated neutralization of HAdV. We observed species-specific neutralization of HAdVs, which is dependent upon defensin binding to the virus capsid. Thousands of defensin molecules bind to each virus particle with an approximate KDifferential susceptibility to defensin was previously observed in studies of cutaneous and genital serotypes of HPV This conclusion is bolstered by the observation that only correctly folded HD5 has antiviral activity. Previous studies showed that the chemokine activity of some defensins is dependent upon defensin conformation max) with an apparent affinity (KD) that approximates the IC50 for infection. The use of surface plasmon resonance to more accurately measure the capsid-defensin interaction would have been preferable for these studies; however, the large mass difference between HD5 (3.6 kDa) and the virus particle (150 MDa) precludes this approach. Although our analysis likely approximates the binding parameters of the system, there are some limitations. First, it is semi-quantitative because of the methods used to estimate both the number of virus particles in each sample and the amount of defensin bound. Second, there is no estimation of non-specific binding. Nonetheless, the data more closely fits a specific binding curve.We used an equilibrium-binding assay to measure the affinity and stoichiometry of the defensin-capsid interaction. We found that as many as 2750 HD5 molecules are bound to each virus particle at saturation  is insufficient to completely abrogate neutralization. Rather, two or more sites must be simultaneously disrupted, as in the PB/GYAR chimera, to generate defensin resistance. Because resistance was also observed in the 19cF construct, at least two separate determinants are likely found in fiber. In each case, disruption of the neutralization sites led not only to resistance, but also to enhancement of infection. This finding supports the notion that enhancement and neutralization are competing processes mediated by defensin binding. Analysis of additional virus chimeras to map the neutralization determinants may provide a more detailed description of the binding sites important for inhibition and enhancement. They may also help explain the resistance of HAdV-41 to HD5 despite the presence of one acidic residue in the identified fiber neutralization determinant of both the short and long HAdV-41 fibers.Based on our combined functional and structural studies, we propose a model for neutralization in which α-defensins bind to critical capsid determinants at the point of contact between fiber and penton base, thereby preventing fiber release. One implication of this model is that fiber dissociation is absolutely required for subsequent uncoating events. This model cannot distinguish between the dissociation of fiber and penton base from the capsid independently or together as a complex. In the first case, defensin may actively lock the fiber onto the penton base. Alternatively, HD5 may obstruct a conformational change in penton base that is required for its release with fiber still attached. Our studies provide strong support for a mechanism of neutralization of HAdV-5 by HD5 and, in combination with our previous report demonstrating stabilization of HAdV-5, -12, and -35 capsids by HD5 Although the temporal order of uncoating events suggests that fiber release is a critical step in vivo against adenovirus or other non-enveloped viruses has not been demonstrated; however, several observations suggest that the neutralization model studied here could be relevant for antiviral immunity. HD5 concentration in the intestinal lumen has been estimated at 14–69 µM (50–250 µg/ml) in vivo.The role of defensins Tissue culture reagents were obtained from Invitrogen . Human A549 cells (ATCC) were propagated in DMEM supplemented with 10% FBS. Stable 293 cells over-expressing the human β5 integrin subunit (293β5) were created by transfecting 293 cells (ATCC) with the human β5 gene . Transfected cells were selected for high integrin expression. Stable 293 cells over-expressing the V-protein of the paramyxovirus Simian virus 5 (293-SV5/V) were a gift of Kenneth Mellits  HAdV-2p, -3p, -4p, -11p, -12p, -25p, -35p, -37p, -41p, and -51p were from ATCC. HAdV-7p and -14p were gifts of David Metzgar . HAdV-16p and -23p were gifts of Adriana Kajon . HAdV-19c was a gift of James Chodosh  Virus chimeras were created by replacing the entire open reading frames of the HAdV-5 penton base or fiber with that of HAdV-19c by recombineering All wild type viruses were propagated in 293β5 or A549 cells except for HAdV-41p, which was propagated in 293-SV5/V. All AdV vectors were propagated in 293β5 cells. Cultures were infected with 300 particles/cell of purified viruses or from cleared lysates of original virus stocks. When complete cytopathic effect was observed, cells were harvested and concentrated by low speed centrifugation. For some serotypes, virus was precipitated from supernatant using 8% PEG Synthetic HNP1, HBD2, and HD5 were obtained from Peptides International, Inc. . HD5 derivatives containing the E14Q substitution or L-α-aminobutyric acid in place of cysteine were produced by solid phase chemical synthesis as described Prior to use in this assay, each virus stock was titrated on A549 cells. A virus concentration was chosen to produce 50–70% maximal signal in the absence of defensin as described below. To measure the effect of defensins on infectivity, purified virus was incubated with HD5 or HNP1 for 1 h on ice in serum-free DMEM (SFM). Confluent A549 cells in black wall, clear bottom 96-well plates were washed twice with SFM, and virus/defensin mixtures were added in a final volume of 35 µl/well. In parallel, wells were infected with two-fold serial dilutions of each virus to establish a standard curve for quantification with an upper limit of 200%. After 2 h, wells were washed twice and replaced with DMEM/10% FBS. Samples were incubated for approximately 48 h, fixed with paraformaldehyde, permeabilized in 20 mM glycine/0.5% Triton X-100 in PBS, and stained with an anti-hexon primary antibody  and an Alexa Fluor 488-conjugated anti-mouse secondary antibody . Plates were scanned for Alexa Fluor 488 signal using a Typhoon Trio variable mode imager . Total well fluorescence above background was quantified with ImageJ software To test the activity of HD5 derivates, Ad5.eGFP was incubated with 15 µM of HD5, HD5-Abu, or HD5-E14Q. Infectivity was assessed by enumerating eGFP-positive cells by flow cytometry as described g (avg.) for 2 hrs at 4°C] on a discontinuous gradient consisting of 300 µl of 30% nycodenz and 200 µl of 80% nycodenz in 50 mM NaCl, 20 mM HEPES pH 7.4 using an SW55ti rotor with adaptors . The visible virus band was collected. The other half of each sample was used to make a standard curve for quantification. All samples were boiled in reducing loading buffer and separated using a 16% PAGEgel  or 10–20% Tris-Tricine gel . The gels were stained with Deep Purple  and imaged on a Typhoon Trio. Virus bands were quantified using ImageQuant NT software . The amount of HD5 in each sample was normalized to protein V and hexon. The amount of HD5 in the centrifuged samples was then quantified against the standard curve using Prism software. Affinity and stoichiometry were estimated from the average data of at least three independent experiments using Prism software.To measure HD5 binding to HAdV-5 and -51, HD5 was serially diluted in PBS and mixed with 5 µg purified virus. After 1 h incubation on ice, one half of each sample was separated by ultracentrifugation [209,000×9 particles/sample) was incubated with or without 20 µM HD5 or HD5-Abu for 45 min on ice. In parallel, 1×105 A549 cells in PBS+0.2% sodium azide were incubated with or without 200 nM 5FK or 16FK for 45 min on ice. The virus/defensin mixtures were combined with the cell/FK mixtures  and incubated for 45 min on ice. Samples were washed 2 times with cold PBS+1% FBS, fixed with 1% paraformaldehyde, and analyzed by flow cytometry for Alexa Fluor 488.Recombinant HAdV-5 fiber knob (5FK) comprising residues 387–581 of the HAdV-5 fiber and HAdV-16 fiber knob (16FK) comprising residues 151–353 of the HAdV-16 fiber, each containing an N-terminal hexahistidine tag, were expressed in BL21(DE3) cells  and purified using TALON Metal Affinity Resin  as previously described 2 and filtered to 12 Å resolution with cosine edge filtering using the BFACTOR program (http://emlab.rose2.brandeis.edu/software).Purified Ad5.F35 (160 µg/ml) was combined with HD5 (20 µM) and incubated for 45 min on ice. CryoEM grids were produced with an FEI Vitrobot. Electron micrographs were collected on an FEI Polara microscope  operated at 300kV with the grids at liquid nitrogen temperature using the SAM semi-automatic data collection routine Difference mapping was performed by docking the HAdV-5 hexon (PDB 1P30) and HAdV-2 penton base/fiber N-terminal peptide crystal structure coordinates (1X9T)


To confirm the presence of Crimean-Congo hemorrhagic fever in Sudan, we tested serum of 8 patients with hemorrhagic fever in a rural hospital in 2008. Reverse transcription–PCR identified Crimean-Congo hemorrhagic fever virus. Its identification as group III lineage indicated links to virus strains from South Africa, Mauritania, and Nigeria. Bunyaviridae, genus Nairovirus) is a tick-borne virus. Its tripartite RNA genome consists of small (S), medium, and large segments. The virus is distributed throughout much of Africa, Asia, and southern Europe  while he was in the hospital, and both acquired the infection . No details were available for 3 other patients with hemorrhagic fever associated with the hospital.Of these 10 patients, 9 were admitted to a rural hospital in Al-fulah, where 6 continued to bleed, subsequently became comatose, and died. Records were unavailable for the other 3. In addition, 3 probable cases in the community were reported. Each of these 3 persons had a course of hemorrhagic disease and death that was compatible with CCHF; they had not been admitted to the hospital and could not be traced because of poor security conditions in the region. Patient ages varied from 15 to 70 years. Nosocomial transmission of the virus was likely the result of lack of personal protection for the hospital staff and the attending relatives, as has been often noted during previous outbreaks  was positive according to reverse transcription–PCR (RT-PCR) specific for CCHFV . The higLaboratory confirmation of 8 cases of CCHF demonstrates the presence of this disease in Sudan. Genetic analysis of the viruses showed that the strain involved was similar to strains found in South Africa, Mauritania, and Nigeria.Detailed analysis of virus outbreaks is often limited by the lack of appropriate high-containment facilities required for virus isolation. However, appropriately sampled and stored acute-phase serum samples can have high titers of the virus, which enable extraction of virus RNA and genome sequencing studies without prior amplification of the virus in cell culture. In this study, serum specimens from 8 patients who died were positive for CCHFV by RT-PCR. Lack of virus-specific antibodies 1–3 days after onset of illness can be explained by the severity and rapid course of the disease, which does not allow sufficient time for antibody production. Virus-specific antibodies are, however, generally seen later in the course of illness and in persons who survive the infection.,Antibody studies have suggested the presence of various arboviruses in Sudan (Clearly, this CCHFV is present in Sudan, and physicians should consider CCHF as a diagnosis for hemorrhagic fever cases in the region. In addition, efforts to provide appropriate personal protective supplies and training to medical staff in rural areas should be increased to help minimize risk for caregivers.


Orthobunyavirus in the family Bunyaviridae. It is transmitted by hematophagous arthropod vectors such as Culicoides biting midges and is widely distributed in temperate to tropical regions of the world. The virus is well known as a teratogenic pathogen which causes abortions, stillbirths, premature births and congenital abnormalities with arthrogryposis-hydranencephaly syndrome in cattle, sheep and goats. On the other hand, it is reported that the virus rarely induces encephalomyelitis in cattle by postnatal infection. A first large-scale epidemic of Akabane viral encephalomyelitis in cattle occurred in the southern part of Japan from summer to autumn in 2006. The aim of this study is to define the epidemiological, pathological and virological properties of the disease.Akabane virus is a member of the genus Nonsuppurative encephalomyelitis was observed in cattle that showed neurological symptoms such as astasia, ataxia, opisthotonus and hypersensitivity in beef and dairy farms by histopathological analysis. Akabane viral antigen and genome were consistently detected from the central nervous system of these animals, and the virus was isolated not only from them but also from the blood samples of clinically healthy calves in the epidemic area. The isolates were classified into genogroup I a containing the Iriki strain, which caused encephalitis of calves almost twenty years ago in Japan. Most of the affected cattle possessed the neutralizing antibody against Akabane virus. Seroconversion of the cohabitated and sentinel cattle in the epidemic area was also confirmed during an outbreak of the disease.The ecological and epidemiological data we have obtained so far demonstrated that the Akabane virus is not endemic in Japan. No evidence of Akabane virus circulation was observed in 2005 through nation-wide serological surveillance, suggesting that a new strain belonging to genogroup I a invaded southern Japan from overseas in the summer of 2006 and caused an unprecedented epizootic of encephalomyelitis mainly in susceptible calves. It will be necessary to reconsider the vaccine strategy to control the disease effectually. Orthobunyavirus in the family Bunyaviridae. It is widely distributed from the tropical to temperate zones of the world and has been associated with hematophagous arthropod vectors such as Culicoides biting midges and mosquitoes, and with ruminants [Akabane virus is classified into the genus uminants -4. Akabauminants -7. Theseuminants ,8,9, anduminants ,11.Orthobunyavirus [It is apparent that Akabane virus shares many common features with other members of the genus nyavirus . It possnyavirus -15. The nyavirus . The S Rnyavirus -19.Aedes vexans and Culex tritaeniorhynchus mosquitoes in Gunma prefecture in 1959 [Culicoides species and cattle in Japan [Since the strain JaGAr 39 (prototype strain of Akabane virus) was first isolated from  in 1959 , many stin Japan -23. Recein Japan ,22,24-27in Japan .In 2006, bovine encephalomyelitis associated with Akabane virus has broken out on a large scale in southern Japan. In this article, we describe the clinical, epidemiological and histopathological features of the disease, and the antigenic and genetic characteristics of the strains isolated from the affected and clinically healthy calves in the epidemic area in order to contribute to the understanding of Akabane viral encephalomyelitis.A total of 180 cattle with neurological disorders were reported in five prefectures  in the southern part of Japan from the end of August to the middle of December in 2006  of 72 cattle with nonsuppurative encephalomyelitis. Finally, seven virus strains were isolated from these PCR-positive specimens by HmLu-1 cell cultures. Four strains were also isolated from the blood or plasma of clinically healthy calves collected between September and October on the affected farms. Furthermore, a strain designated KSB-3/P/06 was isolated from the healthy sentinel cattle in Kyushu Research Station, National Institute of Animal Health, in Kagoshima prefecture during an outbreak of the disease. All 12 strains listed in Table All of the isolates reacted clearly with a monoclonal antibody to the N protein and four of seven neutralizing monoclonal antibodies recognizing epitopes A1, A2, B and C1 on the Gc protein of the Akabane virus OBE-1 strain . The resFurther antigenic comparison between one of the isolates, KM-1/Br/06 strain, and the OBE-1 and Iriki strains was performed by the cross-neutralization test using rabbit antisera to these three strains. As demonstrated by the immunobinding assay, the KM-1/Br/06 strain was antigenically identical to the Iriki strain, but was distant from the OBE-1 strain Table . The OBESerological investigations in the Kumamoto and Kagoshima prefectures indicated that 117 (98.3%) of 119 cattle with neurological symptoms and 113 (74.3%) of 152 cohabitated cattle without neurological symptoms in the case farms possessed the neutralizing antibody against Akabane virus. Annual serological surveillance of arthropod-borne viruses demonstrated that 60 (45.5%) of 132 healthy sentinel calves in both prefectures exhibited seroconversion to Akabane virus between September and November in 2006. No seroconversions to the other bovine arthropod-borne viruses, such as Aino, bluetongue, Chuzan, Ibaraki and bovine ephemeral fever viruses, were observed in these sentinel calves this year.Nucleotide sequences of the S and M RNA segments of isolates were determined, and their open reading frames (ORFs) were compared respectively with those of the Akabane virus strains previously reported . The ORFTo define the genetic relationships between the strains isolated in 2006 and the previous isolates of the Akabane virus, the phylogenetic trees of these two segments were constructed. As previously reported , the fieAn epizootic of the disease with neurological disorders in cattle was observed in the southern part of Japan from summer to autumn in 2006. It was initially suggested that the disease was associated with some neurotropic arthropod-borne viruses due to its seasonal occurrence over an extensive area and to the histopathological findings of the affected cattle. We eventually identified Akabane virus as the cause of the disease, as in that observed in 1984 , throughCulicoides oxystoma is the principal vector and that bovine arthropod-borne viruses have been isolated between summer and autumn (August to November) when adult midges are abundant [Culicoides biting midges carried on the seasonal wind over long distances [Culicoides larvae by vertical transmission should be confirmed by further investigations of virus-vector interactions.Diseases caused by various arthropod-borne viruses, such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever viruses, have been recognized as major obstacles to the production of beef and dairy cattle in Japan. Above all, Akabane virus is considered to be the most important because the virus circulation has been observed almost every year for over four decades since 1959. Seroconversion of sentinel calves has always been confirmed after the rainy season (June to July) through nation-wide serological surveillance of bovine arthropod-borne viruses. Entomological investigations in southern Japan continuing over two decades indicate that abundant . Moleculabundant ,27. Giveistances ,29. A siistances ,26. HoweCulicoides biting midges until late autumn. The virus finally caused an unprecedented outbreak of encephalomyelitis, mainly in susceptible calves. The absence of the virus circulation in 2005 may have increased the number of susceptible animals without antibody against Akabane virus (data not shown). This may be one of the factors that facilitated a large-scale outbreak of the disease.No evidence of Akabane virus circulation was observed in Japan in 2005 via nation-wide surveillance using sentinel animals. Therefore, it is considered that a new strain initially invaded from overseas into Kumamoto prefecture, where the first clinical case was reported on 30 August in 2006, and then was spread throughout the southern part of Japan by There are four Akabane virus genogroups, of which two groups, I and II, are extant in Japan ,27, wherThe KM-1/Br/06 strain was isolated from a 4-day-old calf with nervous symptoms  against the Akabane virus OBE-1 strain [Tissue samples of central nervous systems were collected from 72 affected cattle. These were fixed with 10% buffered formalin and processed for paraffin-wax embedding. The tissue sections from each paraffin block were stained with hematoxylin and eosin for histopathological examination. One pair of serial sections from cattle that showed neurological symptoms was submitted for immunohistochemical assessment of the detection of Akabane viral antigens. After deparaffinization of the pair sections, endogenous peroxidase activity was inhibited by treatment with 3% H1 strain , while tTaq DNA polymerase. The resulting cDNA was amplified by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s followed by one step of final extension at 72°C for 10 min. The PCR products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide.Tissue specimens obtained from the brain and spinal cord of affected cattle were rinsed, minced and homogenized in serum-free Eagle's minimum essential medium (MEM) . The supernatant of a 10% homogenate of each tissue was used for viral RNA preparation. Total RNA was extracted using the QIAamp Viral RNA Mini kit . The primer pairs AKAI172F/AKAI560R (5'-CAGAAGAAGGCCAAGATGGT-3'/5'-AAGTTGACATCCATTCCATC-3') and the QIAGEN OneStep RT-PCR kit (Qiagen) were used for detecting the viral S RNA segment. Reverse transcription was conducted at 50°C for 30 min. This mixture was then heated for 95°C for 15 min to stop the reaction and to activate HotStart The supernatant described above was also used for virus isolation. On the other hand, blood samples were collected from clinically healthy cattle in the affected farms and from sentinel calves. Heparinized blood samples were separated into erythrocytes and plasma by centrifugation, and the erythrocytes were washed three times with cold PBS. The supernatant of 10% homogenates of tissues, plasma and washed erythrocytes were stored at -80°C until use. Several blood samples were directly used as inoculum after being frozen and thawed once. These specimens were inoculated into monolayer cultures of hamster lung (HmLu-1) and/or baby hamster kidney (BHK-21) cells. After incubation at 37°C for 7 days, the cell culture fluids were pooled and subinoculated into freshly prepared cell cultures at least twice until the infected cells exhibited the cytopathic effect.Monoclonal antibodies recognizing the N protein and seven neutralizing epitopes  on the Gc protein of Akabane virus strain OBE-1 were used for antigenic characterization of the isolates . The supBovine sera were serially diluted twofold with serum-free MEM in the 96-well cell culture microplates. Each serum dilution was mixed with an equal volume of 200 50% tissue culture infective doses/0.05 ml of virus and incubated at 37°C for 1 h. Then 0.1 ml volumes of HmLu-1 cells suspended in GIT medium  were added to each well. After incubation at 37°C for 7 days, the antibody titer was determined as a reciprocal of the highest serum dilution showing complete inhibition of the cytopathic effect. For a cross-neutralization test, rabbit immune sera to the OBE-1, Iriki and KM-1/Br/06 strains of Akabane virus were produced by the method described previously  with theViral RNA was extracted from the supernatant of virus-infected cells using the High Pure Viral RNA kit  for sequencing. Oligonucleotide primers AINO-5' (5'-AGTAGTGTGGCTCCAC-3') and AINO-3' (5'-AGTAGTGTACTCCACTAT-3') were used for RT-PCR of the S RNA segment. These primers corresponding to the terminal sequences of the Aino virus S RNA segment are available for specific amplification of the corresponding gene of some other species including Akabane virus ,32. The AB373232, AB373234, AB426271–AB426280 (for the S RNA segment) and AB373233, AB426281–AB426282, AB436954 (for the M RNA segment). The following previously published nucleotide sequence data were used in this study: AB000819, AB000851–AB000854, AB000857, AB000863, AB000864, AB000867, AB232182, AB232191–AB232193, AB232196–AB232198, AB232200, AB232204, AB232207, AB232210, AB232213, AB232214, AB232218, AB232222, AB232231, AB232240, AB232243, AB232251, AB232260, AB232264, AB232265, AB232268, AB232299, AB232314, AB232319, AB232320, M22011 for analysis of the S RNA segment and AB100604, AB100605, AB208700, AB297818–AB297851 for analysis of the M RNA segment.The nucleotide sequence data reported in this article were deposited in the DNA Data Bank of Japan (DDBJ) with the accession numbers RK, MH and SI detected viral genome and isolated viruses from clinical samples of bovine encephalomyelitis, and performed the serological survey. MK, YG and ST carried out histopathological and immunohistochemical investigations. TY contributed to the comparative sequence analysis and the construction of phylogenetic trees of the isolates. TK contributed to the production of rabbit antisera and the antigenic characterization of the isolates. TT participated in the epidemiological analysis of the disease. TI coordinated the experiments and helped to draft the manuscript. MY designed the experiments, analyzed the data and drafted the manuscript. All authors have read and approved the final manuscript.


Mechanism of drug release was found to be swelling and erosion-dependent. The studies showed that formulated alginate and chitosan beads can be used effectively for the delivery of drug to colon and a coat weight of 20% weight gain was sufficient to impart an excellent gastro resistant property to the beads for effective release of drug at higher pH values.The purpose of this research was to develop and evaluate multiparticulates of alginate and chitosan hydrogel beads exploiting pH sensitive property for colon-targeted delivery of theophylline. Alginate and chitosan beads were prepared by ionotropic gelation method followed by enteric coating with Eudragit S100. All formulations were evaluated for particle size, encapsulation efficiency, swellability and  There has been a considerable research for design of colonic drug delivery system. Colon targeting has been achieved by several ways, which include prodrugs, pH- and time-dependent systems. Polysaccharides such as chitosan, pectin, inulin, alginate, guar gum have been explored for their potential for colon specific drug delivery. To achieve successful colon drug delivery, a drug needs to be protected from degradation, release and/or absorption in upper portion of GI tract and then ensure abrupt or controlled release in proximal colon. Colon targeting is usually advised because of advantages i.e, reduced dosing frequency; achieve high concentration at distal gut for local treatment; chronotherapy and delivery of drug to a region that is less hostile metabolically.Multiparticulates offer greater advantages over single unit system as they disperse uniformly in GI tract, offer flexibility and less inter and intra individual variability in formulation process. Multiparticulates present several advantages in comparison to single unit forms that they exhibit higher colonic residence time and more predictable gastric emptying.2+ and Zn2+) or trivalent ions (Al3+), an elastic gel is formed due to ionic interaction between the ions and carboxyl group, mainly of guluronic blocks. Chitosan, a naturally occurring polysaccharide has received major attention in drug delivery system. It is biocompatible and non toxic. Chitosan is weak cationic polysaccharide composed of (a (1→4) 2-amino-2deoxy-β-D-glucan) obtained by alkaline deacetylaion of chitin[Alginate is linear, naturally occurring polysaccharide extracted from brown sea algae. In presence of various divalent  was a gift sample from French Pharma, Chandigarh, India. Chitosan (CH) and sodium alginate were purchased from Sigma Aldrich Chemicals, Bangalore, India. Triethyl citrate was purchased from Himedia laboratories Pvt. Ltd, Mumbai. Ethylcellulose (EC) was purchased from S. D. Fine-Chem Limited, Mumbai, India. Eudragit S100 was a gift sample from Cadila, Ahmedabad, India. All other chemicals used were of analytical reagent grade.Alginate, chitosan beads containing theophylline was prepared by ionotropic gelation method. Theophylline was dispersed in sodium alginate (2% w/v) solution or chitosan (3% w/v) solution in suitable solvents . The druTheophylline was dispersed uniformly in sodium alginate (2% w/v) solution and ethylcellulose (2% w/v) was mixed and stirred for 30 min and then drop wise added to agitated 5% w/v calcium chloride solution 100 ml). Chitosan reinforced alginate beads were prepared by dispersing theophylline in sodium alginate solution (2% w/v). The solution was dropped through a needle (#20) into agitated 5% calcium chloride solutions (100 ml) with chitosan (0.5% w/v) already dissolved in acetic acid solution (1% v/v). Beads were left for 30 min, and washed twice with distilled water and dried at 50° for 4 h and then at room temperature (25°) for 2 days for complete drying0 ml. Chi.% w/v was added and stirring was continued for 30 min. Coating was done with a pan rotating at 20 rpm, with an inlet air temperature 60 to 70° and outlet air temperature of 35 to 40°. The solution was sprayed at an atomizing air pressure of 2 bars. The coating was continued until the desired weight gain was achieved. A series of coated products with different film thickness were produced, quantified by the total weight gain (%TWG), by varying amount of coating solution sprayed[The enteric coating of all the beads bearing theophylline was performed by spray coating in a conventional coating pan. In brief coating solution was prepared by mixing Eudragit S100 with acetone for 1 h using a stirrer. After an hour, triethyl citrate (TEC) 6n sprayed.The particle size of prepared beads was measured with an optical microscope fitted with a calibrated eyepiece. The mean of 50 beads was noted as particle size. To determine the particle size of ethyl cellulose reinforced alginate beads dial thickness meter was used.The theophylline in the beads was determined by digesting the beads in PBS (pH 7.4) and 0.1 N HCl (pH 1.2), respectively, for sodium alginate and chitosan beads. The extracted drug concentration was determined spectrophotometrically  at 272 nm for alginate beads and 269 nm for chitosan beads. The encapsulation efficiency of prepared hydrogel beads was calculated in terms of ratio of drug in formulation to amount of drug added.g–Wo)/Wo×100, where Wo is the initial weight of beads and Wg is the weight of beads in the swelling medium[Swelling studies for coated beads was performed in three different media simulating transition of beads in GIT . Swelling index was calculated from the formula: Swelling index= (Wng medium–14.In vitro drug release studies were performed using USP dissolution test apparatus (Apparatus 2). The dissolution studies were performed in 900 ml of dissolution medium, which was stirred at 100 rpm at 37±0.1° following a pH progression method i.e pH 1.2 for first 2 h, pH 6.8 for next 3 h and pH 7.4 for rest of studies. Aliquots was withdrawn periodically and replaced with fresh medium. Theophylline content in the aliquots was assayed spectrophotometrically  at 269 nm for pH 1.2 and 272 nm for the rest of the samples[e samples.Alginate and chitosan beads were prepared by dropping the solution of the drug alginate dispersion into calcium chloride solution, and for drug-chitosan-dispersion into tripolyphosphate solution, where beads were formed due to cross linking of alginate by calcium ions, and for chitosan with phosphoric ions. Preparation of beads by ionotropic gelation is based on the ability of various polysaccharides such as pectin, alginate, chitosan, and gellan to form a gel in the presence of multivalent ions. Beads can therefore be prepared simply by adding, drop wise, a dispersion of polysaccharide and material to be encapsulated into solution of multivalent ions. The contact of droplets with the ions results in instantaneous formation of spherical gel structures containing the material to be encapsulated uniformly dispersed throughout the polysaccharide matrix.Alginate and chitosan micro beads were successfully prepared by ionotropic gelation method. The beads were coated with Eudragit S100 by spray coating in a conventional coating pan. The coated beads were found to be of spherical shape. Diameter of beads varies as shown in in vitro drug release in simulated GI fluid was studied.Moreover, longer the cross linking time, lower the size of beads. The bead diameter decreased as the cross linking time was increased, because there is greater time for the cross linker to promote formation of cross links with chitosan molecules or alginate molecules and make a more compact mass. The longer the hardening time, the smoother and more spherical were the beads obtained. Coating thickness over drug bearing beads was obtained in terms of total weight gain (TWG) of beads after enteric coating with Eudragit S100 dispersion and their effect on The effect of various formulations parameters on entrapment efficiency of prepared hydrogel beads are shown in 2+ and Na+ exchange. In initial phase Na+ ions present in phosphate buffer exchange with Ca2+ ions, bound to COO- groups of mannuronic blocks. As a result, an electrostatic repulsion between negatively charged COO- groups increases resulting in gel swelling. Exchanged Ca2+ ions precipitate in the form of insoluble calcium phosphate reflecting in slight turbidity of swelling medium. In the later phase of swelling, diffusion of Ca2+ from polyguluronate blocks cause loosening of tight egg-box structure and thus permit the penetration of additional amounts of media into the beads[Swelling studies for coated beads was performed in three different pH  simulating transition of beads in GIT. Swelling studies carried out shows that maximum swelling for all batches of beads is in simulated intestinal fluid (pH 7.4) as shown in figs. the beads.In case of chitosan beads, ionization of amine group on chitosan decreases with increasing pH proceeding to weaker cross linking density. At higher pH open porous structure with low density were also reported. In caseIn case of uncoated beads, nearly >70% of drug was released in initial 4 h of dissolution study. This situation is not acceptable for the drugs that are required to be released in colon. Therefore beads were coated with Eudragit S100 polymer to retard the release of drug until the pH reaches above 7.0.In vitro release studies were carried out in order to evaluate effect of Eudragit S100 coat with an objective to release most of drug in the colon. The initial release of TH from coated hydrogel beads was low. However, some part of drug (10 to 15%) was released in initial 2 h from the beads having TWG 10%. In case of TWG 20%, very less amount of drug was found to be released (3 to 4%) which is less as compared to drug release from TWG 10%. Figs. g 10 to 1% was relIn case of alginate beads  having a coat of TWG 10%, at the end of 5 h the release was found to be 26.25±1.09, 6.80±1.81, 10.58±1.29, 11.07±1.59 and 6.07±2.35%, respectively. In case of beads having a coat of TWG 20%, Eudragit S100 coat release at end of 5 h was found to be 7.18±1.19, 4.51±1.23, 3.29±1.15, 4.23±1.29 and 2.66±1.89%, respectively. For chitosan beads  having a coat of TWG 10% Eudragit S100 coat, release at end of 5 h was found to be 26.68±1.33, 18.06±1.28, 13.25±1.17 and 6.28±1.09%, respectively. In case of beads having a coat of TWG 20% Eudragit S100 coat release at the end of 5 h was found to be 3.59±1.63, 1.27±1.09, 6.73±1.13 and 2.54±1.47%, respectively.However, >80% of drug was released when pH was gradually raised above 7.0. The result indicates that drug was protected completely from upper GIT condition by a coating of Eudragit S100, because Eudragit polymer contain carboxyl group that ionize in an environment where pH is greater then 7. As ionization take place, integrity of film was disturbed and drug is released. At pH 7.4, membrane coating get dissolved and beads were exposed to dissolution media following which the polymer matrix swells and erodes releasing entrapped drug.2 = 0.8471–0.9591) as compared to zero order (R2 = 0.7022–0.8560) for formulations having TWG 10% coat thickness. Formulations having TWG 20% coat thickness also showed the linearity with respect to first order (R2 = 0.8833–0.9545) as compared to zero order (R2 = 0.7188–0.8090). Higuchi's equations for formulation having TWG 10% coat thickness ranges from (R2 = 0.7101–0.8278) and for formulation having TWG 20% coat thickness ranges from (R2 = 0.71936–0.7879) hence to confirm precisely the domination mechanism, the data was plotted according to Korsemeyer's equation. The resultant ‘n’ values was >1, for the all coated beads, indicating the super-case II transport mechanism.Drug release data was plotted according to first order, Higuchi's and Korsemeyer-Peppas equation to know the release mechanisms. The formulations showed linearity with respect to first order (RResults of release studies indicate that Eudragit S100 coated hydrogel beads offer a high degree of protection from premature drug release in simulated upper GIT conditions. A well coat of Eudragit S100 delivers almost intact beads to colon, an envi


Emergence of this virus is a serious medical, veterinary, and public health challenge. A recent Hendra virus outbreak at a veterinary clinic in Brisbane, Queensland, Australia, involved 5 equine and 2 human infections. In contrast to previous outbreaks, infected horses had predominantly encephalitic, rather than respiratory, signs. After an incubation period of 9–16 days, influenza-like illnesses developed in the 2 persons before progressing to encephalitis; 1 died. Both patients were given ribavirin. Basal serum and cerebrospinal fluid levels were 10–13 mg/L after intravenous administration and 6 mg/L after oral administration (isolate 90% inhibitory concentration 64 mg/L). Both patients were exposed to infected horses, 1 during the late incubation period in a horse. The attack rate for veterinary clinic staff exposed to infected horses was 10%. An isolate from this outbreak showed genetic heterogeneity with isolates from a concurrent, but geographically remote, outbreak and from previous outbreaks. Emergence of Hendra virus is a serious medical, veterinary, and public health challenge. Henipavirus of the family Paramyxoviridae contains 2 recently described viruses, Hendra virus and Nipah virus, whose natural reservoir is fruit bats of the genus Pteropus  in Thornlands, Queensland, was quarantined after 2 acutely ill horses were provisionally diagnosed with Hendra virus infection. Four horses eventually died from the infection, and another was humanely killed after it recovered, in accordance with established national veterinary practice (9 cells/L) and thrombocytopenia (79 × 109 cells/L); a normal chest radiograph; and negative PCR results for respiratory viruses, including influenza, on a nasopharyngeal aspirate (NPA) specimen. Hendra virus RNA was detected by reverse transcription–PCR (RT-PCR) in serum and NPA specimens. The patient remained clinically well and showed defervescence on day 4 of illness.A 33-year-old man (equine veterinarian) at the clinic had a 2-day history of an ILI  in mid-July 2008. Clinical examination showed only a fever (38°C); mild neutropenia  showed bilateral high-voltage slow waves but no epileptiform activity. He was treated with intravenous ribavirin ; enteric aspirin (100 mg/day); and because of progressive confusion and ataxia, intravenous dexamethasone (4 mg every 6 h).By day 5, mild confusion, ataxia, bilateral ptosis, but no other neurologic signs, developed. Magnetic resonance imaging (MRI) showed multifocal  hyperintense lesions on T2 flair images associated with evidence of infarction on diffusion weighted images. A cerebrospinal fluid (CSF) sample had a leukocyte count of 4 × 106 cells/L, a protein level of 650 mg/L, and a glucose level of 4.3 mmol/L; Hendra virus RNA was not detected by RT-PCR. He died on day 40 of illness. A postmortem examination was not performed because a risk assessment conducted by Queensland Health concluded that an examination could not be performed in Queensland.Over the next 4 days (to day 10) progressive neurologic signs and high-grade fever developed, culminating in a generalized partial tonic-clonic seizure. During the next 4 weeks, the patient required mechanical ventilation, remained febrile, and despite control of seizure activity and minimal sedation, remained deeply unconscious with minimally reactive pupils. Treatment with ribavirin was stopped after 12 days of therapy when the patient’s hemoglobin level decreased to 76 g/L. Serial MRI showed marked progression with innumerable cortical, subcortical, and brainstem hyperintense foci on T2 flair images with matching diffusion restriction, and leptomeningeal enhancement , panel AA 21-year-old woman (veterinary nurse) at the same clinic was observed 4 days after patient 1 with a 3-day history of an ILI. Results of clinical examination were unremarkable apart from a fever (39°C). Results of initial investigations, including complete blood examination, chest radiography, and EEG, were normal. Hendra virus RNA was detected in serum and NPA specimens. She was treated with intravenous ribavirin  and aspirin (100 mg/day). On day 12 of illness, 96 hours after defervescence, encephalitic manifestations  developed. Results of MRI and a CSF specimen were normal; Hendra virus was not detected by RT-PCR.Over the next 12 days, signs of encephalitis deteriorated  before stabilizing. Serial MRI showed scattered, small, hyperintense, cortical and subcortical foci on T2 flair images and leptomeningeal enhancement , panel BTreatment with ribavirin was continued at a dosage of 600 mg orally every 8 h , and she remained clinically well during 20 weeks of follow-up. Repeat MRI (at days 69 and 135 and month 11) showed marked resolution in signal abnormalities, and EEGs (at days 69 and 182) also showed considerable improvement. A CSF sample obtained at month 12 showed normal results, including a negative result for Hendra virus by RT-PCR.Serial quantitative RT-PCR for Hendra virus RNA . SeroconDirect PCR sequencing of clinical material from both patients was used to obtain nucleotide sequences for the nucleoprotein, matrix, glycoprotein, and accessory protein genes and the intragenic junctions. Results showed that Hendra virus associated with this outbreak was distinct from Hendra virus in a concurrent equine outbreak in Cannonvale in northern Queensland, Australia, and from viruses in previous outbreaks .Virus isolated from Vero E6 cells  cultured with an NPA sample obtained on day 5 of illness of patient 2 was used to test in vitro susceptibility to ribavirin (>2 weeks after last exposure to infected horses or humans). One veterinary worker had percutaneous blood exposure while performing euthanasia on the Hendra virus–infected horse that recovered. This person was treated with a 5-day course of intravenous ribavirin  within 4 h after exposure. Serologic follow-up up to 25 weeks showed no evidence of infection with Hendra virus.After confirmation of equine Hendra virus cases, 83 contacts of infected horses  and 8 contacts of human case-patients  were contacted for assessment and counseling. Nonspecific respiratory or gastrointestinal symptoms developed in 16 contacts of infected horses; none had Hendra virus RNA or antibodies against this virus in convalescent-phase serum samples  were assessed in 28 veterinary clinic staff. Twenty (including the 2 patients) reported contact with potentially infected equine body fluids; the Hendra virus attack rate for exposed staff was 10%. Fourteen identified high-risk exposures , among whom self-reported use of PPE  was low (7%). The other 6 staff identified low-risk exposures (intact skin). High-risk exposures identified among the 2 infected patients included performing daily nasal cavity lavage during the last 3 days of the incubation period on 1 infected horse  and participating in a necropsy of another infected horse (patient 1) 16 days before illness onset.Unlike previously recognized equine Hendra virus infections   and basal serum ribavirin concentrations of 6 mg/L  were documented, these concentrations may be inadequate given the results of in vitro susceptibility testing. The 90% inhibitory concentration of ribavirin against one of the human virus isolates was 64 mg/L, which was similar to that reported for an original human isolate from 1994  for patient 2 but later  in patient 1. However, the influence of this timing on outcome remains unclear, particularly given the uncertainty about activity of ribavirin against Hendra virus and other possible confounding factors. For example, patient 1 had different and possibly greater quantitative initial exposure to Hendra virus, earlier onset of encephalitis, and corticosteroid therapy that may have suppressed virus clearance. We also administered intravenous ribavirin prophylactically for 5 days in another person after a percutaneous blood exposure to a horse documented as positive for Hendra virus by RT-PCR. This regimen was well tolerated, and follow-up assessments have not demonstrated seroconversion.We emphasize that the efficacy of ribavirin as therapy or prophylaxis remains, at best, uncertain. However, given the absence of other available therapies, and the tolerability of ribavirin, we advocate that it be given early in clinical illness and be considered where high-risk exposure to Hendra virus has occurred. However, because only 6 cases have been recognized in humans, the criteria for high-risk exposure remain undefined.This absence of established therapies underscores the need for early recognition of equine Hendra virus infection, implementation of infection control precautions, and prevention of horse-to-human transmission. Although the attack rate for humans exposed to infected horses in this outbreak (10%) was similar to that in the other documented stable outbreak that caused human infection (at which time the causative pathogen had not been identified) .The modes of horse-to-human transmission of Hendra virus are not known completely, but likely result from direct contact with respiratory secretions and other equine tissue and fluids or from droplet or aerosol exposure. Furthermore, difficulties associated with clinical recognition of equine Hendra virus infection present challenging infection control implications for equine veterinary practice. Therefore, routine adoption of infection control precautions encompassing hand washing, use of gloves and other forms of PPE, and a high index of clinical suspicion for equine Hendra virus infection are required. Although experience with human Hendra virus infection is relatively limited, no evidence exists of human-to-human transmission. However, persistence of Hendra virus RNA in NPA samples throughout the first 2 weeks of illness indicates that healthcare workers potentially exposed to respiratory secretions should adopt PPE use to prevent exposure to droplets.In conclusion, we describe severe Hendra virus infection in 2 veterinary workers, 1 who died, after exposure to infected horses. Given the absence of other established therapies, ribavirin was administered. However, further understanding of the pathogenesis of neurologic disease is required to guide potential therapies. These evolving equine clinical manifestations and the inevitable and increasing interactions between the natural host of the virus (fruit bat species) and horses will continue to challenge veterinary, public health, and medical communities.Virologic and serologic results for 2 patients with Hendra virus encephalitis, Australia, 2008*


We recently determined that Nanjianyin virus, isolated from serum of a patient in Yunnan Province, China, in 1989, is a type of Kyasanur Forest disease virus. Results of a 1987–1990 seroepidemiologic investigation in Yunnan Province had shown that residents of the Hengduan Mountain region had been infected with Nanjianyin virus. Flavivirus, family Flaviviridae, can cause fever, hemorrhage, and encephalitis and has a 3%–5% case-fatality ratio  virus, a member of the tick-borne encephalitis virus serocomplex of the genus th India .Flavivirus. No further tests to classify Nanjianyin virus were performed at the time it was isolated. The virus was preserved by lyophilization and stored at –30°C.In tests conducted shortly after isolation of Nanjianyin virus in 1989, the virus caused a typical cytopathic effect within 4 days after its injection in BHK-21 cells, killed 100% of 3-day-old mice within 2.5 days after their intracerebral inoculation with a 25-μL culture supernatant, and killed 100% of 50-day-old adult mice within 11–13 days of their intraperitoneal inoculation with a 30-μL culture supernatant. Hemagglutination inhibition test results showing a cross-reaction between Nanjinayin virus and a Japanese encephalitis virus antibody indicated that Nanjianyin virus belonged to the genus g. We then extracted the total RNA from 140 µL of supernatant by using the QIAamp Viral RNA Mini Kit  in accordance with the manufacturer’s protocol and produced the first strands of cDNA by using Ready-To-Go You-Prime First-Strand Beads  as described in the manual accompanying the kit. We used Flavivirus genus-specific primers (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the nucleotide DNA sequence of Nanjianyin virus was 99% homologous to that of KFD virus .Recently, we used molecular methods to determine that Nanjianyin virus is a variant of KFD virus. After reconstituting the lyophilized virus in a BioSafety Level 3 biosafety cabinet, we suspended the sample in 0.5 mL minimum essential media  (pH 7.4) and then centrifuged it for 5 min at 6,000× To complete the sequence determination of the PrM-E genes, we designed 3 pairs of primers to amplify them. Using information from a previous study .Results of sequence alignment and homology analysis performed with MegAlign software of DNASTAR  showed that the 654-bp PrM gene of Nanjianyin virus was 99.6% identical to that of KFD virus (Itp9605 strain), 99.4% identical to that of KFD virus (EU480489), 98.2% identical to that of KFD virus (X74111), but only 90.4% identical to that of Alkhurma hemorrhagic fever (AFH) virus (1176 strain), and only 57.2% to 64.3% identical to the 654-bp PrM genes of other tick-borne encephalitis complex viruses such as Omsk hemorrhagic fever virus (Kubrin strain), tick-borne encephalitis virus (Sengzhang strain), Powassan virus (LB strain), and Langat virus (TP21 strain). The 1487-bp E gene nucleotide sequence of Nanjianyin virus was 99.8% identical to that of KFD virus (Itp9605 strain), 99.8% identical to that of KFD virus (EU480489), 98.5.0% identical to that of KFD virus (X74111), 91.9% identical to that of AFH virus (1176 strain), and <72% identical to that of other tick-borne encephalitis complex viruses. The nucleotide sequence of the 1,000-bp NS5 gene of Nanjianyin virus was 99.6%, 99.7%, and 99.7% homologous to that of KFD virus (Itp9605 strain), KFD virus (W371), and KFD virus (EU480489), respectively; 92.3% homologous to that of AFH virus isolate 1176; and <77.6% homologous to the 1,000-bp NS5 gene of other tick-borne encephalitis complex viruses. Results of homology analyses thus demonstrated that Nanjianyin virus belongs to the KFD virus clade, and results of phylogenetic analyses conducted with 2,142 nt of the PrM-E gene and 1,000 nt of the NS5 gene suggested that Nanjianyin virus and KFD virus are in the same genetic cluster .Macaca mulatta) in the Hengduan Mountain region (Lushui and Eryuan counties) (Results of a serosurvey of tick-borne viruses conducted from 1987 through 1990 in Yunnan Province (Haemaphysalis spinigera, which is the vector for KFD virus in the region (Results of epidemiologic and virologic investigations suggest that migratory birds play a key role in the spread of arboviruses (In summary, we found that Nanjianyin virus, first isolated in the Hengduan Mountain region of Yunnan Province, is a variant of KFD virus. This finding confirms that infection with KFD virus has previously occurred in the region and justifies enhanced surveillance for KFD among febrile patients in the Hengduan Mountain region.


Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons.Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by ≥2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage.In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood. Distinct genetic populations of HIV-1 in the genital tract compared to blood have been reported in 170 men The central role of the female genital tract in both sexual and perinatal HIV-1 transmission underscores the importance of understanding viral evolution within these tissues, which should contribute to the development of effective treatments and prevention strategies, including vaccines. While multiple reports suggest that HIV-1 appears compartmentalized between the genital tract and the blood of women Studies reporting compartmentalization of viruses within the female genital tract have often analyzed only cell-free viruses A cross-sectional study of chronically HIV-1 infected women compared viral populations in each woman's genital tract to her blood. Viral sequences were derived by single genome amplification (SGA). To control for variations in HIV-1 shedding throughout the menstrual cycle subjects' study visits were conducted during the luteal phase of their cycle Study eligibility was not limited by use of antiretroviral drugs. Participants were classified into one of three categories according to antiretroviral treatment (ART) and plasma viral load at the time specimens were collected: “effective ART”, when virus replication was suppressed to <50 copies/mL; “failing ART”, defined as receiving ART, but with plasma HIV-1 RNA >400 c/mL; and “no ART”, when not receiving ART.The study was conducted at University of Washington, following procedures approved by the University of Washington Institutional Review Board. All study participants provided written informed consent.Blood plasma and PBMC were separated using Accuspin™ tubes . Cells were counted using a Beckman Coulter Z1 Coulter Particle Counter  or hemocytometer. The cell pellet and plasma fraction were stored at −80°C until nucleic acids were extracted.os. After absorption of ∼24 uL of fluid the strips were visually inspected for blood contamination and placed in guanidinium isothiocyanate extraction solution  os was obtained using Baby Tischler Cervical Biopsy Forceps  and placed into RPMI. The cervical tissues were snap-frozen in Optimal Cutting Temperature freezing medium  by floating in a cryomold (Tissue-Tek) on top of 2-methyl-butane cooled by dry ice. Tissues were stored at −80°C until sectioned for DNA extraction.Cervical secretions were obtained by placing three filter paper wicks  into the cervical Virions in 500 µL of plasma were concentrated by ultra-centrifugation. Guanidinium isothiocyanate extraction solution  was added to viral pellet. GeneAmplimer pAW 109 RNA plasmid  was added to plasma virus or 300 µL of cervical secretions in guanidinium solution. Silica was used to extract nucleic acids DNA was extracted from PBMC using the IsoQuick nucleic acid extraction kit . Cervical DNA was isolated from 4 to 8 sections, each 20 µM thick, of the punch biopsy tissue, using the Gentra DNA Purification System . Viral genome of extracted DNA and some cDNA samples were quantified using limiting dilution PCR envenv was conducted as described below. None of these controls yielded amplicons, suggesting negligible DNA contamination in plasma and Sno-strip specimens from these subjects. Viral gene sequences corresponding to the C2-V5 region of HIV-1 env were derived as previously described env was chosen for its genetic diversity and because it amplifies in multiplexed end-point dilution PCR at a sensitivity similar to regions encoding RT and PR The nucleic acids in 0.2–1 mL of plasma and 20–50 µL of genital secretions were extracted using either the Qiagen QIAamp Viral RNA Mini-kit  or when the viral load was below the limit of detection (<50 copies/mL) using silica with slight modifications Sequences were assembled and checked for read errors and hypermutation nn) compares genetic distances between sequences within and between tissues independent of phylogeny b compares the number of nodes between sequences within and between tissues; and (4) the Critchlow coefficient r compares tree-based genetic distances within and between tissues nn and correlation coefficients were calculated in HyPhy with 1,000 permutations between tissues to determine statistical significance The compartmental structure of viral sequences from each subject was evaluated by four distinct statistical tests using either tree- or distance-based parameters: (1) Slatkin and Maddison (SM) evaluates variation from normalcy in the distribution of sequences over a predicted tree structure using MacClade Divergence of each sequence from the MRCA and population diversity (estimated as the average pair-wise genetic distances) were calculated using PAUP* version 4.0b10 with the corresponding evolutionary model. The mean diversity and divergence were calculated for each subject's anatomical sites .Non-synonymous and synonymous distances  and dN/dS ratios were based on maximum likelihood trees with codon substitution models  env region in the Viral Epidemiology Signature Pattern Analysis program (http://www.hiv.lanl.gov/content/hiv-db/P-vespa/vespa.html) http://hiv-web.lanl.gov/content/hiv-db/ENTROPY/entropy.html).Atypical amino acid residues in genital tract sequences compared with peripheral blood sequences were evaluated using the aligned protein sequences from the C2-V5 http://hiv-web.lanl.gov/content/hiv-db/GLYCOSITE/glycosite.html). HIV-1 co-receptor usage was predicted for the V3 region amino acid sequences using the position-specific scoring X4/R5 and SI/NSI matrices (subtype B sequences), and/or the presence of basic amino acids at V3 region sites 11 or 25  http://indra.mullins.microbiol.washington.edu/pssm/).The number of potential N-linked glycosylation sites in blood and genital tract sequences was determined using the N-GLYCOSITE program nn, and Critchlow correlations, was evaluated using GraphPad software and expressed as a κ−score .Confidence intervals for entropy, average dN/dS values, and mean diversity values were calculated using JMP . Viral divergence values, number of dN/dS sites and entropy between sequence sources  were compared using Wilcoxon Rank Sums tests . Diversity values were calculated using the mean pair-wise distances for all sequence comparisons from a tissue DNA sequences derived from biopsied cervical tissue could be from infected genital tract cells or from cells circulating in blood vessels within the cervical tissue. Pair-wise distances generated in PAUP were used to identify whether the nearest neighbor to each genital DNA sequence was another genital tract or a blood sequence. For each patient, generalized estimating equations (GEE) with an exchangeable correlation matrix was used to calculate 95% confidence intervals for the proportion of pair-wise comparisons where genital DNA sequences were nearest to other genital RNA and/or DNA sequences using SAS v9.1 . Under the assumption of random matching, we calculated the random probability (proportion) that any sequence would be matched with a genital DNA or genital RNA sequence [/]. If the random proportion fell outside of the GEE-based confidence intervals, we concluded there was statistically significant evidence that genital DNA sequences were more likely from the cervix rather than a contaminant from blood passing through cervical blood vessels.The gene sequences determined in this study were deposited in GenBank under accession numbers EF624488-EF625226.b and r (κ score = 0.68).Cross-sectional analyses of HIV-1 populations in the genital tract and blood were conducted on thirteen subjects . A mediaClose inspection of the five subjects with statistical evidence of compartmentalization by three or more tests revealed pairs and small clades of genetically similar and/or monotypic  viruses from the genital tract and/or blood  We recognize that cell-associated viral sequences derived from genital specimens could potentially include sequences from blood due to HIV-1 infected cells traversing through blood vessels in the cervix. To evaluate this possibility we assumed that if genital cell-associated sequences were actually from blood, their distribution within the phylogram should mirror blood. Visual examination of the phylograms reveal in most subjects that genital tract DNA sequences cluster in large part with other genital RNA or DNA sequences –4. NotNo signature amino acid residues were identified in HIV-1 sequences from the genital tract or peripheral blood of the 13 participants (data not shown). Sites undergoing positive selection were found in all 13 subjects. However, none were associated with compartmentalization of virus between the blood and genital tract  in the blood of three  and in the genital tract of two women . Overall, no trend was noted for N-linked glycosylation sites in genital compared to blood viruses (p = 0.59 Wilcoxon Rank Sum Test).CCR5 was predicted to be the predominant co-receptor for virus in all of the subjects studied. HIV-1 viruses from three women were also predicted to use the CXCR4 co-receptor, including viruses from both the genital tract and peripheral blood. The CCR5- and CXCR4-using viruses clustered separately in phylogenetic trees , which should sample the entire cervical and vaginal surfaces, and presumably represent the most fit variant Phylogenetic analyses found tissue-specific clades with strong bootstrap support in half of the participants. However, in all but two women small clades of viruses comprised of blood or genital tract sequences intermingled in the phylograms. Across the subjects, each genital clade with >70% bootstrap support includes viruses with little genetic diversity suggestive of a burst of viral replication similar to that observed for viruses from macaque cervices env populations from uterine cervix and blood. Across our subjects we did not observe tissue-specific signature amino acid residues, differential co-receptor usage, or evidence for differential immune selection (as measured by the patterns of non-synonymous site mutations) in either genital tract or blood viruses. Viral population entropy and diversity are also comparable in the genital tract and peripheral blood of subjects we and others Further support for mixing of genital and blood HIV-1 comes from additional comparisons of the HIV-1 Taken together, our data suggest that when statistical analyses of HIV-1 detect compartmentalization of genital tract and blood viruses in cross-sectional studies it is often due to low diversity or monotypic clusters, typical of a burst of replication or proliferation of a cell with provirus within the genital tract. The interspersed arrangement of genital and blood clades across the phylograms of most subjects we studied questions whether the genital population in each woman is distinct from viral population in her blood.Across the literature, compartmentalization of HIV-1 within the genital tract of women by statistical measures and detection of discordant patterns of drug-resistance has been interpreted as evolution of independent viral populations within the genital tract env sequences analyzed were often low diversity (<1%) cell-free virus that is consistent with recent rounds of viral replication env sequences clustered by the sampling time as well as tissue type Previous studies eliminated identical sequences from the analysis Two longitudinal studies of drug resistance mutations in genital tract secretions and blood detected mutational discordance from one study visit that resolved at the next study visit A potential difference between our study and those focused on cell-free virions centers on the observation that approximately 33% of 311 women in situ viruses and not blood contaminates similar to others' observations A limitation of our study is the uncertainty that genital HIV-1 DNA and RNA indeed originate from the genital tract, and are not due to blood contamination of the cervical biopsies or Sno-strip samples. Several experimental controls and analyses suggest that the majority of sequences we derived from the genital tract were likely from In summary, our analyses of viral sequences from the blood and genital tract of women with chronic HIV-1 infection suggest that the multiple clusters of genetically similar and monotypic viruses, particularly from the cervix, are likely from recent viral replication or proliferation of cells with provirus, respectively. Statistical tests used to assess virus compartmentalization appear biased by these tissue-specific low-diversity viruses over the intermingling of blood and genital clades evident in phylograms. If our hypothesis is correct, then few if any significant barriers exist to the flow of HIV-1 between the female genital tract and blood, and unique genital tract viral lineages should not evolve and persist over time, which is a hallmark of a viral compartment Table S1Sites with Positive Selection and Entropy Differences(0.43 MB TIF)Click here for additional data file.


Sir,vs intravenously administered therapies. In particular, this paper compares Uftoral® (tegafur–uracil), with capecitabine, based on unit costs for drugs as outlined in the British National Formulary, 2002. However, there are a number of discrepancies we would like to highlight with this assessment. First, as of March 2006 the price of Uftoral® (tegafur–uracil) was reduced as indicated in the following table:With reference to the study by et al is replicated and we include the new NHS price for Uftoral®, the treatment costs for a 12-week course of tegafur with uracil would be £1788. This is lower than any of the other therapies referred to in the article and represents a cost saving vs capecitabine of £343.1 over a 12-week period.Given this significant change in price, over a standard three cycles of treatment (105 days), the new price represents a 59% reduction in drug costs (including costs of folinic acid). If the cost minimisation assessment as completed by Ward et al report the frequency of consultations and hospitalisations relating to adverse events over the treatment period for capecitabine, UFT/LV  and the Mayo regimens. The sources referenced for UFT/LV are et al, is not reported in this publication. Additionally, we believe the reference, ‘‘Xeloda (capecitabine): achieving clinical excellence in the treatment of metastatic colorectal cancer. Roche submission to the National Institute of Clinical Excellence’, to be either incorrect or inappropriate as this source cannot be referenced. Therefore, we question whether the stated figures for UFT/LV for, ‘A and E’, ‘Clinic Consultation’ and ‘Other hospital visits’ are appropriate for inclusion in such an analysis. We also dispute why these categories would have a zero frequency of incidence for patients treated with capecitabine, yet be an issue with UFT/LV.We believe that there are also inaccuracies in the estimation of costs related to adverse events included within the model. In table 5, Ward Finally, we would also dispute how the adverse event costs for Uftoral® are assumed to be equal to that of the Mayo regimen. We believe that this is an overestimation and biased against Uftoral®. We would propose that adverse event costs for Uftoral® are more likely to be lower or at least equal to capecitabine, given the incidence of hand and foot syndrome as a prominent adverse event for capecitabine.et al, misrepresents the current costs of treating metastatic colorectal cancer with Uftoral® and this could result in clinicians being less willing to include Uftoral® in their treatment protocols.Given the points raised, the article by Ward


Although arboviral infections including Chikungunya virus (CHIKV) are common in sub-Saharan Africa, data on their circulation and prevalence are poorly documented. In 2006, more than 400 cases of dengue-like fever were reported in Kumbo (Northwest Region of Cameroon). The aim of this study was to identify the aetiology of this fever and to define its extent in the area.We conducted a cross-sectional seroprevalence survey one year after clinical investigations to define the extent of the infection. An entomological survey consisted of the collection and identification of mosquito immature stages in water containers in or around human dwellings.Aedes africanus and the remaining Culex spp.A total of 105 sera were obtained from volunteers and tested for CHIKV, O'Nyong-nyong virus (ONNV) and Dengue virus (DENV) specific IgM and IgG antibodies by enzyme-linked immunosorbent assays (ELISA). CHIKV infection was defined as the presence of IgM antibodies to CHIKV. There was serological evidence for recent Chikungunya infection, as 54 subjects (51.4%) had detectable IgM anti-CHIKV in their sera. Amongst these, 52 showed both anti-CHIKV IgM and IgG, and 2 (1.9%) had IgM anti-CHIKV in the absence of IgG. Isolated anti-CHIKV IgG positives were detected in 41 (39%) cases. No anti-ONNV and anti-DENV IgM antibodies were found amongst the sample tested. Out of 305 larvae collected in the different breeding sites, 87 developed to the adult stage; 56 (64.4%) were Ae. africanus which was found to be relatively abundant among the raffia palm bushes probably plays a role in the transmission of CHIKV along the chain of sylvatic/domestic mosquito species in this rural area. Particular attention should therefore be given to arbovirus infections in the Central African sub-region where these infections are becoming an emerging public health threat.These findings suggest that the outbreak of febrile illness reported in three villages of Western Cameroon was due to CHIKV. The issue of a possible persistence of anti-CHIKV IgM antibodies is discussed.  Alphavirus genus (Togaviridae family) and belongs to the Semliki virus antigenic complex comprising species with similar clinical manifestations and close antigenic patterns like CHIKV, O'Nyong-nyong virus (ONNV), Ross River virus and Mayaro virus [Chikungunya virus (CHIKV), is a member of the ro virus ,2.Aedes spp. mosquitoes. The virus has been isolated from sylvatic mosquito species in several countries including Senegal, Côte d'Ivoire, Central African Republic and South Africa. The mosquito species involved vary geographically and with ecological conditions; however, the major species involved in sylvatic cycles are Ae. furcifer, Ae. taylori, Ae. luteocephalus, Ae. africanus and Ae. neoafricanus [CHIKV is now known to circulate in enzootic cycles throughout much of sub-Saharan Africa where it originated. In West and Central Africa, the disease is maintained in a sylvatic cycle involving wild non-human primates and forest-dwelling fricanus -5. In rufricanus .Ae. aegypti and Ae. albopictus with humans and mosquitoes as the only hosts of the virus [In Asian and African urban cycles, the disease is transmitted by he virus ,8. CHIKVhe virus  but alsohe virus -13 leadihe virus .In Cameroon, cases of CHIKV were reported in 2006 in the two main cities of the country: Douala and Yaoundé . That saThis paper describes the findings of the serological and entomological surveys conducted from November 18-26, 2007, at the beginning of the dry season in three villages of Kumbo. Results of such a study are invaluable for public health systems in Central African region where epidemics are frequently underreported or misreported.Investigations were carried out in three villages, Ngehndzen, Ndzeru and Tasaï where clinical cases were registered during the outbreak in 2006.These villages belong to the Mbam health care area and are situated about 30 km north-east of the centre of Kumbo Figure . They alWe conducted a cross-sectional seroprevalence study one year after clinical investigations. A week before the investigation, a local health care worker went round the villages to make the population aware of the study. At our arrival, informed consent was obtained from volunteers gathered at the meeting points  before blood collection. As the study was considered by the MOH as part of its public health response to the outbreak (letter number D112-212/NS/MSP/SG/CNEC/SP), no authorization was required from the national ethics committee.Aedes spp mosquitoes. Larvae and pupae were collected and reared until the adult stage which was identified using morphological keys.During the entomological survey oral consent of householders was requested for inspection of their houses. Emphasis was put on households where cases of acute febrile syndrome were reported the previous year. Indoor and outdoor water storage (i.e. domestic), abandoned (i.e. peridomestic) containers, and natural receptacles and pools containing water were inspected in human dwellings and among raffia bushes for evidence of the presence of immature stages of Sera were separated from whole blood specimens and tested for IgM and IgG antibodies to CHIKV and ONNV in the Reference Centre for Arboviruses, Lyon (France), and antibodies to DENV in the laboratory of virology (Centre Pasteur Cameroon) using "in-house" techniques such as IgM capture enzyme immunoassay (MAC-ELISA) and ELISA IgG. For MAC-ELISA, IgM antibodies were captured with a goat anti-human IgM antibody . CHIKV, ONNV or DENV antigens were then added. Specific binding was demonstrated by using a CHIKV, ONNV or DENV mouse hyper immune ascitic fluid and a goat anti-mouse peroxidase-labelled conjugate . For the detection of IgG, microplates were coated with specific antigens  and a specific binding was demonstrated by using a peroxidase-labelled goat anti-human IgG conjugate.Sera were considered positive when the optical density of the assay exceeded the mean optical density of negative controls plus three standard deviations.2 was used for statistical testing and the level of statistical significance chosen for all analyses was p < 0.05.Results of serological analysis were entered and analyzed using Epi Info 2002 software (Centre for Disease Control and Prevention). Age, sex, and locality distributions of the seropositive participants were compared. χA total of 105 persons (sex ratio m/f 0.44) were sampled for blood collection of whom 95 reported suffering clinical manifestations of a dengue-like syndrome during the outbreak period (about one year before this survey). The mean age was 50 years  with a standard deviation of 17.5. Most of the people examined were farmers (85.8%).There was serological evidence for recent Chikungunya infection, as 54 subjects 51.4%) had detectable IgM anti-CHIKV in their sera  that were positive for anti-CHIKV IgG antibodies were also positive for anti-ONNV IgG antibodies due to the cross reactivity occurring between CHIKV and ONNV. No antibodies to DENV were detected.IgM seropositivity to CHIKV was similar between females (52%) and males (50%) Table . BecauseSeropositivity to CHIKV was similar in the different villages Table .Aedes africanus and the remaining Culex spp. Moreover, a number of adults of Ae. africanus mosquitoes were identified biting in raffia bushes during the day and this was consistent with complaints by the local populations of a high burden of mosquito bites in this particular biotope. Although only 3 breeding sites were found with culicine larvae in this environment, it was evident that places where water collected among raffia bushes probably represented important breeding places for Ae. africanus immature stages.During the entomological survey 78 householders gave their oral consent for inspection of their houses. Most of them reported suffering the acute febrile syndrome the previous year. Eighteen to 30 households of 3 to 6 houses were surveyed in each village. All water containers, in or around human dwellings were inspected for the presence of mosquito larvae. Culicine larvae were found in 4 peridomestic breeding sites  outside human dwellings and in 3 locations situated in raffia bushes where water had collected. Out of 305 larvae collected in these different breeding sites, 87 developed to the adult stage; 56 (64.4%) were The prevalence of anti-CHIKV IgM antibodies one year after the outbreak was high and similar to data recorded during previous outbreaks of CHIKV ,17. NeveAlthough we did not collect blood samples from unaffected areas (control) for comparison purposes, the possibility of the persistence of IgM anti-CHIKV for one year cannot be ruled out. This apparent persistence of anti-CHIKV IgM is consistent with the observations of both Fourie & Morrison and Gauzere ,20 who dPlasmodium falciparum infection and/or lack of diagnostic tools in local health care centres. Thus only a few sporadic cases of arboviral infections have been reported in recent years across Cameroon [Although arboviral infections including CHIKV are common in sub-Saharan Africa ,16,23-26Cameroon ,22,27. HAedes spp bites either in their dwellings or in the raffia bushes where they fetch firewood (women), collect palm wine (men) and water (children).The outbreak affected equally the three villages in Kumbo , and both sexes, and all age groups. Despite the over-representation of women (sex-ratio 0.44) among the persons sampled during this survey, both sexes exhibited the same seropositivity rates. The possible explanation to this is that women, men and children are equally exposed to Despite the fact that the extensive serologic overlap occurring between CHIKV and ONNV precludes any definitive interpretation of the results as to which virus may have been responsible for the production of the IgG antibodies in 90.5% of the sera , the oveAe. albopictus into Africa [Ae. albopictus was identified as the primary vector in the Chikungunya outbreak in Libreville (Gabon) [Ae. albopictus or Ae. aegypti, were found during this survey probably because the study took place during the dry season which is probably not suitable for their emergence. Moreover, these two species were described in the North-West regional capital (about a hundred km to the south) during the rainy season [As the CHIKV outbreak in Kumbo and Yaoundé/Douala occurred during the same period, it is possible that the same virus strain was implicated. It has been shown that the Cameroon strain is highly similar to the CHIKV strain reported in the sub-region including Democratic Republic of Congo and Gabon . Howevero Africa . Indeed o Africa ,33. Ae.  (Gabon) ,35 and w (Gabon) . This in (Gabon) -38. HoweAe. africanus could at least be involved in the transmission of CHIKV down the chain of sylvatic/domestic mosquito species in this area. It is possible that by entering areas like raffia bushes, and so being in contact with the sylvatic Ae. Africanus, humans might have become infected therefore serving as their incidental hosts. These humans might have then provided a source of the virus to infect potential peridomestic mosquitoes , which then became involved in the transmission cycle of the virus. CHIKV is known to circulate in a natural cycle amongst forest-associated simians and sylvatic mosquito species in West and Central African jungles, spasmodically causing outbreaks of varying size and intensity amongst the local populations living near the jungles [Although we did not search for the virus in mosquitoes collected,  jungles ,40.Ae. africanus which was found to be relatively abundant in the raffia bushes could be involved in the transmission of CHIKV along the chain of sylvatic/domestic mosquito species in this rural area via humans and simians that come into contact with the virus either in the gallery forest or raffia bushes surrounding the villages.Between April and July 2006, CHIKV was identified during an outbreak of a febrile syndrome in Yaoundé and Douala . In the This study, alongside previous ones, provides further evidence of the circulation of the CHIKV in the Cameroon forest region. Despite the co-endemicity of malaria and arbovirus infections in the Central African sub-region CHIKV infection is an emerging public health threat which should be given particular attention. In the absence of a vaccine, more effort should be put into prevention by involving the local population in vector control activities.The authors declare that they have no competing interests.MD and EN conceived and designed the study. CAN carried out entomologic investigations. HZ performed CHIKV serologic assays. MD and CP analysed the data. MD wrote the initial draft and CAN, DR and JCM revised the manuscript. All authors read and approved the final manuscript.MD is a young virologist in charge of the newly created Arbovirus and Haemorrhagic Fever virus branch in the Laboratory of Virology at Centre Pasteur Cameroon. Since 2006, his research interests have focused on diagnosis and epidemiology of arbovirus diseases .