Orthobunyavirus genus in the family Bunyaviridae. Genetic analyses of ORO-like strains from Iquitos, Peru, identified a novel reassortant containing the S and L segments of ORO virus and the M segment of a novel Simbu serogroup virus. This new pathogen, which we named Iquitos (IQT) virus, was first isolated during 1999 from a febrile patient in Iquitos, an Amazonian city in Peru. Subsequently, the virus was identified as the cause of outbreaks of “Oropouche fever” during 2005 and 2006 in Iquitos. In addition to the identification of 17 isolates of IQT virus between 1999 and 2006, surveys for neutralizing antibody among Iquitos residents revealed prevalence rates of 14.9% for ORO virus and 15.4% for IQT virus. Limited studies indicate that prior infection with ORO virus does not seem to protect against disease caused with the IQT virus infection. Identification of a new Orthobunyavirus human pathogen in the Amazon region of Peru highlights the need for strengthening surveillance activities and laboratory capabilities, and investigating the emergence of new pathogens in tropical regions of South America.Oropouche (ORO) virus, a member of the Simbu serogroup, is one of the few human pathogens in the  Orthobunyavirus genus in the family Bunyaviridae. Phylogenetic analyses of ORO-like strains isolated from febrile patients in Iquitos, Peru, identified a novel ORO reassortant virus, which we named Iquitos (IQT) virus based on the location of the isolation of the virus. This novel pathogen was first isolated during 1999 from a 13-year-old boy who had an illness that included symptoms of fever, headache, eye pain, body pain, arthralgias, diarrhea, and chills. Subsequently, the virus was identified as the cause of outbreaks of “Oropouche fever” during 2005 and 2006 in Iquitos. Limited serological studies indicate that prior infection with ORO virus does not seem to protect against disease caused with the IQT virus infection. In summary, we identified a new Orthobunyavirus human pathogen in the Amazon region of Peru; these results highlight the need for strengthening surveillance activities and investigating the emergence of new pathogens in tropical regions of South America.Oropouche (ORO) virus is one of the few human pathogens in the  Bunyaviridae are classified into five genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospoviruses. The orthobunyaviruses are enveloped, negative sense RNA viruses whose genome comprises three segments: small (S), medium (M) and large (L). The S segment encodes for the nucleocapsid and a nonstructural protein, NSs. The M segment encodes for the glycoproteins Gn and Gc, whereas the L segment encodes the viral polymerase. Some members of the Orthobunyavirus genus are known to cause clinical disease in humans, including Oropouche (ORO) virus, a member of the Simbu serogroup, which causes a febrile disease often associated with headache, dizziness, weakness, myalgias and arthralgias Viruses in the family Orthobunyavirus genus occurs in nature and has led to the emergence of new viruses, occasionally with increased pathogenicity. This appears to be the case with Ngari virus, which has been associated with hemorrhagic fever in Kenya and Somalia Genetic reassortment among members of the same serogroup within the Orthobunyavirus genus, 18 serogroups have been recognized on the basis of the results of cross-hemagglutination inhibition (HAI) and antibody neutralization relationships Within the Coquillettidia venezuelensis in Trinidad Ochlerotatus serratus and Culex quinquefasciatus in Brazil Culicoides paraensisC. paraensis, from infected to susceptible hamsters, and from infected humans to susceptible hamsters Bradypus tridactylusCallithrix sp.) in Brazil ORO virus has been isolated from mosquitoes  in Lima, in collaboration with the Ministry of Health of Peru, initiated a passive surveillance study to investigate etiology of febrile illnesses. As part of these surveillance activities, several ORO-like strains were obtained from febrile patients in Iquitos, Peru. In this study, we describe the identification of a novel reassortant virus which we named Iquitos (IQT) based on the location of the isolation of the virus. Specifically, we: 1) demonstrated that IQT virus causes clinical disease in humans similar to ORO fever, 2) described the genetic relationship of IQT virus to other members of the Simbu serogroup, 3) identified the risk factors associated with human infection by IQT virus in an urban setting of the Amazon region of Peru, and 4) describe the clinical manifestations associated with infection. Significantly, immunity to ORO virus does not appear to protect against infection by IQT virus, and both ORO and IQT viruses appear to have similar antibody prevalence in Iquitos over the past 10 years.The IQT1690 ORO strain used in this study was isolated in 1995 from a 50 year-old male resident of Iquitos, Peru. The first strain of IQT virus (IQT9924) was isolated in Iquitos from a 13 year-old boy . The oriIquitos is a city of about 380,000 inhabitants located 120 meters above sea level in the Amazon Basin in northeastern Peru.The human use study protocols were approved by the Ministries of Health of Peru and by the NAMRU-6 Institutional Review Board (protocol NMRCD.2000.0006). The study subjects were patients (≥ 5 years of age) who presented with a diagnosis of an acute, febrile undifferentiated illness at military or civilian outpatient clinics in Iquitos. The criteria for inclusion in the program was fever ≥ 38°C of no more than five days duration, headache, myalgia and other nonspecific symptoms. Demographic and clinical information were obtained from each patient at the time of voluntary enrollment and a signed consent form was obtained from patients 18 years of age and older. In addition, written assent was obtained from patients between 8 and 17 years of age. For patients younger than 18 years, written consent was obtained from parents or legal guardians. Paired-blood samples were collected, one during the acute phase of illness and a second sample 2-4 weeks after onset of symptoms.Acute serum samples were tested for virus by cell culture, and both acute and convalescent serum samples were assayed for IgM antibody to a variety of arboviruses by an enzyme-linked immunosorbent assay (ELISA), as described previously Aedes albopictus mosquito (C6/36) cells. Vero and C6/36 cell cultures were maintained at 37°C and 28°C, respectively, and were examined daily for 10 days for evidence of viral cytopathic effects (CPE). Spot-slides of C6/36 and Vero cells were subsequently prepared on days 10 post-inoculation of the samples (or sooner if CPE developed) and an immunofluorescence assay (IFA) was performed using polyclonal antibody against arboviruses endemic to Peru, including ORO virus For virus isolation attempts, serum samples were diluted 1∶5 in Eagle's minimum essential medium (EMEM) supplemented with 2% fetal bovine serum, 200 µg of streptomycin, and 200U/ml of penicillin. Two hundred µl of the diluted samples were inoculated into flasks containing either confluent monolayers of African green monkey kidney (Vero) cells or A total of 1037 human serum samples collected in Iquitos during 2006 were tested for IgG antibody to the ORO strain IQT1690 and IQT9924 virus using a previously described ELISA 2, 2.5 units of GoTaq DNA polymerase  and 5 µl of cDNA. The conditions for the PCRs included incubation at 94°C for 2 minutes, 35 cycles of 94°C for 30 seconds, 50°C for 1 minute, 72°C for 1.5 minutes and a final extension of 72°C for 10 minutes to ensure complete double-stranded DNA synthesis. The primers used for the PCR amplification have been previously described and included ORO N3 (GTGAATTCCCACTATATGCCAATTCCGAATT) and ORO N5 (AAAGAGGATCCAATAATGTCAGAGTTCATTT) that amplifies the S segment, M14C (CGG AAT TCA GTA GTG TAC TACC) and M619R (GAC ATA TG (CT) TGA TTG AAG CAA GCA TG) that amplifies the M segment and M13CBunL1C (TGTAAAACGACGGCCAGTAGTGTACTCCT), and BunL605R (AGTGAAGTCICCATGTGC) that amplifies the L segment Viral RNA was extracted using the QIAamp viral RNA mini kit  or Trizol reagent  following the manufacturer's protocols. The reverse transcription reaction (RT) was done using 1X RT buffer, 0.2 mM dNTPs, 1 µM of primers, 80 units of RNAsin ribonuclease inhibitor , 1mM of dithiothreitol, 200U of SuperScript reverse transcriptase (Invitrogen) and 5 µl of RNA. The reactions were incubated at 42°C for 1 hour. The PCR included 1X PCR buffer, 0.25 mM dNTPs, 1 µM of primers, 3 mM of MgClPartial sequences of the S, M, and L segments were obtained and compared to published ORO virus sequences. Purified PCR products were sequenced directly and sequencing analyses of the PCR products were performed using an Applied Biosystems Prism automated DNA sequencing kit  according to the manufacturer’s’ protocols. Sequences were aligned using the Clustal program in the Mac Vector software package  and phylogenetic analyses were performed using the maximum parsimony, neighbor joining, and maximum likelihood methods implemented in the PAUP software To prepare ORO virus hyperimmune ascitic fluid for classical cross-neutralization tests, mice received 4 weekly intraperitoneal (IP) injections of virus-infected newborn mouse brain suspension with Freud's adjuvant. To investigate antigenic differences among the viruses, cross-neutralization assays were then performed, using a previously described PRNT 50 was calculated by the Reed and Muench method Three to 4 week-old golden Syrian female hamsters were inoculated (IP) with serial 10-fold dilutions of virus. The LDTo determine viremia levels in infected patients, serum samples were prepared in 10-fold dilutions and 100 µl of each dilution was inoculated onto confluent monolayers of Vero cells in 12 well-plates. Viruses were adsorbed to the monolayers for 1 hour at 37°C. A 3-ml overlay consisting of EMEM with 0.4% agarose was added, and the cells were incubated at 37°C for 72 hours. Agar plugs were removed, and the cells were stained with 0.25% crystal violet in 20% methanol. The sensitivity of the assay corresponded to a detection limit of 100 PFU/ml.The analysis was performed using SPSS 17.0 for Windows . The proportions of positive results (PRNT for IQT1690 and IQT9924 antibody ≥ 20) were calculated with their respective 95% confidence intervals (95% CI). The proportions were compared using the Pearson Chi-Square and Fisher exact tests. A 2-tailed p-values < 0.05 was used for all statistical analyses. Multivariate analysis was performed using logistic regression (enter method) adjusting by gender, age , occupation, type of house, contact with domestic animals, travel history and neighborhoods.In 1999, IQT virus strain IQT9924 was isolated in Iquitos, Peru, from a 13-year-old boy who had an illness that included symptoms of fever, headache, eye pain, body pain, arthralgias, diarrhea, and chills. These clinical symptoms were typical of ORO virus infections in Peru Sixteen additional IQT9924 virus isolates  were obtained from febrile patients living in Iquitos: 1 in 2003, 7 in 2005, and 8 in 2006. Of the 17 IQT 9924 virus positive patients, detailed clinical information was available for 16 . The mosBased on virus isolation and serological analyses, the peak incidence for ORO virus was reported in Iquitos as occurring in 2004, 2005 and 2006 Genetic analyses of the S, M, and L segments were conducted to determine the relationships of the isolates to ORO virus and other members of the Simbu serogroup. Phylogenetic trees were constructed for both the S- and M-RNA segments based on members of the Simbu serogroup that included an example from each of the three known ORO genotypes We investigated the serological relationships of IQT9924 virus using cross-neutralization tests. Mouse antisera were prepared against ORO virus strain Trinidad55, IQT9924 virus, and JAT virus. These antisera displayed a 4-fold or greater difference in neutralization titer among these viruses , indicatNext, convalescent sera from patients in Iquitos who had been infected with either OROV or IQT9924 virus were assayed by PRNT to confirm the antigenic differences. A 4-fold or greater difference in neutralization titer was observed between these viruses  corrobor50 of 4220 PFU and was similar to two Panamanian strains (GML 444477 and GML445252) belonging to ORO genotype III that were non-lethal at the highest doses inoculated  .The hamster is used as an animal model of ORO virus infection ctively) . In comp3 and 7×105 PFU/ml whereas 17 patients infected with IQT9924 virus had levels that were below the limit of detection of the assay (<100) to 1.8×105 PFU/ml .Examination of 1037 human serum samples from acute febrile infections in Iquitos revealed that the overall neutralizing antibody prevalence to ORO virus was 14.9% (154/1037) (95% CI 12.8–17.1) whereas prevalence to IQT9924 virus was 15.4% (160/1037) (95% CI 13.3–17.7). Only 3.4% (35/1037) of the serum samples had neutralizing antibodies (>20) to both viruses. Neutralizing antibody prevalence to both ORO and IQT9924 viruses was higher among females than males . Neutralizing antibody prevalence to both viruses in the study population increased with age after adulthood (0% in 5–9 years old to 32.3% in 40-49 years old for ORO virus and 3.6% in 5–9 years old to 25% in >70 years old for IQT9924 virus)  and Bellavista Nanay (16.5%) was higher compared to other neighborhoods . Similar4 virus) . The antLogistic regression models were used to test the significance of each factor. The variables predictive of ORO virus neutralizing antibody prevalence in the model were gender, age and neighborhood, whereas factors such as gender, age, neighborhood, and contact with pigs were predictive of IQT9924 virus antibody prevalence.The first confirmed ORO fever cases in Peru were reported in 1992 involving a small outbreak of eight febrile cases living in Iquitos 10 suckling mice LD50 (SMLD50)/ml among ORO- infected patients, including approximately 10% of the patients who developed viremia titers ranging from 5.0 to 5.3 log10 SMLD50/ml during the first two days of illness C. paraensis and transmission was demonstrated to susceptible hosts. Consequently, it has been postulated that humans are the primary amplifying host during ORO fever epidemics 10 SMLD50/ml were not sufficient to infect C. paraensis50) and found that the average viremia levels detected among ORO and IQT9924 virus-infected patients were 4.8±0.7 log10 PFU/ml and 3.4±1.1 log10 PFU/ml, respectively with at least one ORO-infected patient developing a viremia titer of 5.9 log10 PFU/ml  as high as 35% in certain neighborhoods near Iquitos Our clinical data indicate that the IQT9924 virus shares many of the same clinical manifestations as ORO virus. Headache and chills affected the vast majority of IQT9924-infected patients, similar to previous studies of ORO virus infection Orthobunyavirus genus, studies have shown that the pathogenicity of these viruses is multigenic with the M segment being a major determinant Among the Orthobunyavirus member of the Simbu-serogroup because there have been cases of disease caused by this virus in the Iquitos area over several years. While reassortment among members of the same serogroup of the Bunyaviridae has been identified, few reassortants have been associated with human disease. It will be important to determine the geographic distribution of Iquitos virus and to evaluate its potential as a major public health problem as ORO virus has been done in Brazil.In summary, this study identified a new pathogen, IQT9924 a reassortant of ORO virus that was associated with febrile illness in the Amazon region of Peru. We propose the name Iquitos virus for this newly identified


The 15th International Conference on Human Retrovirology: HTLV and Related Retroviruses was organized in Belgium in the cities of Leuven and Gembloux from June 5 to June 8, 2011. The main topics of the meeting were human T-lymphotropic virus (HTLV), Xenotropic murine leukemia virus-related virus (XMRV) and endogenous retroviruses. The following abstracts were presented.


Interventions are needed to reduce human exposure to hantaviruses. >1 hantaviruses; the highest prevalence was among agricultural workers. Because of the high level of human exposure to hantavirus strains and the severity of resulting disease, additional studies are warranted to determine the reservoirs, ecologic range, and public health effect of this novel strain of hantavirus.To better describe the genetic diversity of hantaviruses associated with human illness in South America, we screened blood samples from febrile patients in Chapare Province in central Bolivia during 2008–2009 for recent hantavirus infection. Hantavirus RNA was detected in 3 patients, including 1 who died. Partial RNA sequences of small and medium segments from the 3 patients were most closely related to Andes virus lineages but distinct (<90% nt identity) from reported strains. A survey for IgG against hantaviruses among residents of Chapare Province indicated that 12.2% of the population had past exposure to  Bunyaviridae, genus Hantavirus) are trisegmented negative-strand RNA viruses in which the small (S), medium (M), and large (L) genomic segments encode for the nucleocapsid protein (N), 2 envelope glycoproteins (Gn and Gc), and the viral polymerase, respectively. Hantaviruses are maintained in rodent reservoirs, and human exposure typically results from inhalation of aerosols from infectious urine or feces, although human-to-human transmission of Andes virus (ANDV) has also been described   as reservoir host of LNV in Bolivia  of a novel strain of hantavirus in 3 patients, including 1 who died. In addition, we report results of a survey to determine the prevalence of previous hantavirus exposure in the region.<7 days) at the Hospital San Francisco de Asis or Centro de Salud Eterezama , located in the Chapare Province of the Department of Cochabamba in central Bolivia  were also approved by the US Naval Medical Research Unit Institutional Review Board in compliance with all US Federal regulations governing the protection of human subjects. Written consent was obtained from patients >18 years of age) were invited to participate in the study. Blood samples (10 mL) were collected by venipuncture for screening of antibodies against hantaviruses, and demographic data were collected for risk factor analysis in assorted villages in Chapare Province  were added, and plates were incubated for 1 h at 37°C. Wells were subsequently incubated with ANDV or LNV antigen for 1 h at 37°C. Viral antigens were recognized by the addition of hyperimmune mouse ascitic fluid for 1 h at 37°C and incubation with horseradish peroxidase–conjugated anti-mouse IgG for 1 h. Finally, substrate  was added, and optical density at a wavelength of 405 nm was measured by using a spectrophotometer. Patient serum specimens were also screened for IgM against a panel of arboviral pathogens, including dengue viruses, yellow fever virus, and Venezuelan equine encephalitis virus. Virus culture and identification was attempted in African green monkey Vero cell cultures by indirect immunofluorescence assay and Sin Nombre virus (SNV) polyclonal antibodies, as described for arboviruses . Serum samples were diluted 1:100 and incubated in SNV recombinant antigen–coated wells and then with horseradish peroxidase–conjugated mouse anti-human IgG . Finally, substrate  was added, and absorbance was measured at 405 nm with a Dynex ELISA MRX Revelation absorbance reader . Samples with optical densities greater than the mean of 5 negative controls plus 5 SD at a 1:100 dilution were considered positive.After serologic screening, RNA was extracted from whole blood and serum samples of patients positive for hantavirus IgM by using the QIAamp Viral RNA Mini Kit . A 1-step reverse transcription PCR (RT-PCR) was performed by using the Access RT-PCR system . Nested PCRs were performed by using the FastStart PCR Master . Initial screening was performed by using primers specific for the S segment as described , RIOMV strain HTN-007 , SNV strain NMH10 , El Moro Canyon virus strain RM97 , Choclo virus , Caño Delgadito virus , Pergamino virus , ANDV strain AH-1 , ANDV strain CHI7913 , ANDV strain Chile-9717869 , Maciel virus strain 13796 , Catacamas virus , Paranoa virus (S: EF576661), Oran virus , LNV , BMJV , Lechiguanas virus strain 22819 , ANDV strain Hu39694 , Playa de Oro virus , Neembucu virus (S: DQ345763), Alta Paraguay virus (S: DQ345762), Itapua virus (S: DQ345765), Araraquara virus , Araucaria virus strain HPR/03–99 (S: AY740630), Jabora virus strain Akp8048 (S: JN232080), Juquitiba virus strain Olfo_777 (S: GU213198), and Castelo dos Sonhos virus .S segment and M segment sequences (submitted to GenBank under accession nos. JF750417–JF750422) were compared with sequences from other members of the genus www.clustal.org) with manual adjustments, and pairwise genetic distances were calculated by using MEGA4.0  were male patients with a median age of 31 years (range 7–95 years). IgM against ANDV (n = 8) or LNV (n = 1) antigen was identified in acute-phase or convalescent-phase samples from 9 (2.4%) patients. No evidence of recent arbovirus infection was detected in these samples. Of the 9 patients with IgM against hantaviruses, 7 (77.8%) were male patients with a median age of 32 years (range 15–49 years). Three of the 9 patients were positive for hantavirus RNA.During January 2008–June 2009, serum samples from 372 febrile patients reporting to clinics in Chapare Province, Bolivia  were tesPatient 1 (FVB0554) was an 18-year-old man (student) from the town of Pedro Domingo Murillo, Bolivia, who came to Hospital San Francisco de Asis in January 2008 He reported 7 days of fevers, chills, and malaise. Other symptoms included oliguria, arthralgias, myalgias, bone pain, headache, and retroocular pain. Gastrointestinal  and respiratory  manifestations were also prominent. The patient died the next day. IgM against LNV antigen  was detected in a serum sample collected before his death.Patient 2 (FVB0640) was a 27-year-old man  from Samuzabety, Bolivia, who came to Hospital San Francisco de Asis in March 2008. The patient had a temperature of 39.9°C and reported 8 days of fever, chills, and malaise. Other symptoms included cough, arthralgias, myalgias, bone pain, headache, and retroocular pain. On examination, multiple cutaneous manifestations were noted, including petechiae, purpura, a maculopapular rash, and a diffuse erythematous rash. The patient was hospitalized for 4 days and recovered. IgM against ANDV was detected in an acute-phase serum sample ; no convalescent-phase sample was obtained.Patient 3 (FVB0799) was a 49-year-old man (farmer) from Flor de San Pedro, Bolivia, who came to Hospital San Francisco de Asis in June 2009. He reported 4 days of fever, chills, and malaise. Other symptoms included arthralgias, myalgias, bone pain, abdominal pain, headache, cough and dyspnea. The patient survived. IgM against ANDV was detected in an acute-phase serum sample ; no convalescent-phase sample was available for additional analysis.Andes virus  and >99.2% pairwise nucleotide identity in the M segment (1–10-nt differences). Nucleotide sequences were compared with those of hantavirus strains available in GenBank . In paires virus .In pairwise comparisons of M segment gene sequences, the highest nucleotide identity (83.3%) was observed in comparison with CASV. Similar amino acid identities were observed with CASV (95.1%), Oran virus (95.3%), Lechiguanas virus (95.0%), and ANDV Hu39694 (95.3%) . Viral swww.ncbi.nlm.nih.gov/ICTVdb/index.htm); formed a clade with ARAV, MACV, PRGV, and other ANDV strains; and formed a subclade with CASV  had IgG against SNV antigen , and thewww.ictvdb.org/Ictv/index.htm), which include a >7% difference in amino acid identity in comparison with previously identified complete S segment and M segment gene sequences, a >4-fold difference in results of 2-way cross-neutralization tests, and occupation of a unique ecologic niche, such as a different primary rodent reservoir. As with other hantavirus strains, attempts to isolate virus in tissue culture were unsuccessful; thus, cross-neutralization studies could not be conducted. Genetic criteria, amino acid and nucleotide comparisons, and phylogenetic analysis clearly support inclusion of this strain in the species Andes virus.We demonstrated the association of a novel genotype of ANDV with fatal human infection in central Bolivia and extended the known genetic diversity of hantaviruses circulating in South America. One fatal case occurred among the 3 patients described, which was consistent with high mortality rates observed with infections with ANDV lineages in neighboring Brazil and Argentina  in Bolivia , Necromys spp. (MACV and ARAV), and Bolomys spp. (MACV) rodents. Potential reservoir species are abundant in Bolivia, including Oligoryzomys spp., Akodon spp., and Calomys spp. (LNV) rodents. Increased rodent population density has been associated with the emergence of hantavirus infection in humans (In this study, we made no effort to incriminate the reservoir host for TUNV. The only hantavirus reservoir identified in South America is rodents of the subfamily


In southern Europe, Toscana virus is one of the three leading causes of aseptic meningitis. Phlebotomus perniciosus in central Italy. Many case reports in travelers and clinical research and epidemiologic studies conducted around the Mediterranean region have shown that TOSV has a tropism for the central nervous system (CNS) and is a major cause of meningitis and encephalitis in countries in which it circulates. In central Italy, TOSV is the most frequent cause of meningitis from May to October, far exceeding enteroviruses. In other northern Mediterranean countries, TOSV is among the 3 most prevalent viruses associated with meningitis during the warm seasons. Therefore, TOSV must be considered an emerging pathogen. Here, we review the epidemiology of TOSV in Europe and determine questions that should be addressed in future studies. Despite increasing evidence of its major role in medicine as an emerging cause of CNS infections, TOSV remains an unstudied pathogen, and few physicians are aware of its potential to cause CNS infections.Toscana virus (TOSV) is an arthropodborne virus first identified in 1971 from the sandfly  Phlebotomus perniciosus collected in Monte Argentario   was originally isolated in 1971 from the sandfly Phlebovirus in the family Bunyaviridae. TOSV is an arthropodborne virus. The lack of biochemical and genetic data for most phleboviruses dictates that the species are defined by serologic relationships and are distinguishable by 4-fold differences in 2-way neutralization tests. Phleboviruses contain a negative-sense, single-stranded RNA genome that consists of 3 segments, designated large, medium, and small, which encode the RNA-dependent RNA polymerase, the envelope glycoproteins, and the nucleoprotein, respectively.According the 8th report of the International Committee on Taxonomy of Viruses, TOSV is a serotype of Sandfly fever Naples virus within the genus Phlebotomos papatasi) distribution. Until recent years, the known distribution of TOSV was limited to Italy and Portugal  of a Swedish patient who was returning to his home country from Portugal. More recently, the geographic distribution of the virus has been extended to France, Spain, Slovenia, Greece, Cyprus, and Turkey, according to results from viral isolation and serologic surveys (–Phlebotomus (sandfly) fever viruses have been isolated from sandflies in southern Europe, Africa, central Asia, and the Americas, and evidence exists for the presence of different viruses in the same sandfly population. Sandfly fever Naples (but not the TOSV serotype) and Sicilian viruses have the widest geographic distribution, in parallel to their vector's   , and in 0.7% by enzyme immunoassay (EIA). In 2,034 German patients, who were hospitalized for various diseases, 1.6% were positive for anti-TOSV IgG by IFA, and 0.8% by EIA. Anti-TOSV IgG was detected in 43 samples of commercial immunoglobulins at titers of 10–1,000 by EIA. Although the seroprevalence of antibodies to TOSV is low in Germany, TOSV infection should be considered in patients returning from virus-endemic areas who have fever and headaches or symptoms of meningitis   with headache , fever (76%–97%), nausea and vomiting (67%–88%), and myalgias (18%). Physical examination may show neck rigidity (53%–95%), Kernig signs (87%), poor levels of consciousness (12%), tremors (2.6%), paresis (1.7%), and nystagmus (5.2%) . In most cases reported so far, CSF contained >5–10 cells with normoglycorachia and normoproteinorachia. Blood samples may show leukocytosis (29%) or leukopenia (6%). The mean duration of the disease is 7 days, and the outcome is usually favorable.,Although TOSV infection in most cases consists of a mild disease with a favorable outcome, a small number of severe cases have been reported in the literature. Two young brothers and a sister living in Umbria experienced TOSV infection in the form of severe meningoencephalitis with stiff neck, deep coma, maculopapular rash, diffuse lymphadenopathy, hepatosplenomegaly, renal involvement, skin rash with lamellar desquamation, a tendency to bleed, and diffuse intravascular coagulopathy. CNS manifestations occurred after 3 weeks of fever. Convalescence was marked by hydrocephalus that required a ventriculoatrial shunt. Diagnosis was established by serologic means and by PCR sequencing  collected in 2 localities of the Tuscany region of Italy between 1980 and 1985. Thirty-seven (44%) were identified as TOSV and 47 (56%) as a new member of the phlebotomus fever serogroup, Arbia virus. The overall virus isolation rate from sandflies was 0.5%. Viral isolation rates for both viruses were similar in different years and in the 2 localities, suggesting that the 2 virus types were active in the sandfly population simultaneously (maximum activity in July) . Similar results were described in a study on some variants in the N gene of strains isolated from humans from 1995 to 1998; only 1 variant showed a single amino acid substitution of an 80-amino-acid region  and low viremia titers. However, ≈30 cases of viral transmission were documented in 2002 and 2003 in the United States and Canada, as well as cases of West Nile virus infections after organ transplantation from a viremic donor. Moreover, 540 positive blood donation samples were detected by using PCR, which underlines the necessity of this kind of test in an epidemiologic situation similar to that seen in the United States. Recent data on TOSV circulation in Mediterranean countries during the summer raise concerns about potential implications for blood donations.Limited studies have been conducted on the genetic variability of TOSV. The work of Sanchez-Seco on the L segment demonstrated the presence of 2 geographically distinct populations of the virus (


In contrast, the small-plaque variants of the virus and the variants isolated from the brain of mice that were infected with the parental virus VN1194 had much higher virulence in mice. The virulence attenuation of serially propagated virus may be caused by the reduced neurotropism in mice. Our whole genome sequence analysis revealed substitutions of a total of two amino acids in PB1, three in PB2, two in PA common for virulence attenuated variants, all or part of which may be correlated with the virulence attenuation and reduced neurotropism of the serially propagated VN1194 in mice. Our study indicates that serial passages of VN1194  H5N1 avian influenza virus continues to cause human infections with high mortality, posing a serious pandemic threat to human health. Although human-to-human transmission of the virus has not yet been very efficient  up to noA/Vietnam/1194/2004 is a clade 1 H5N1 influenza virus strain originally isolated from a fatal human case in 2004. It is also highly pathogenic to mice, which is one of mammalian infection models to determine the virulence and pathogenic mechanism of H5N1 influenza virus ,13-16. TIn this current study, we investigated the differences between the wild A/Vietnam/1194/2004 virus and the serially propagated virus in plaque forming, virulence and genome sequences. We further determined the attenuation of the serially propagated virus in mouse model and its potential mechanisms.in vitro had been propagated for one time in MDCK cells (35°C). All of the experiments with live H5N1 viruses were done in a bio-safety level three containment laboratory approved by the Biosafety Management Committee of State Key Laboratory of Pathogens and Bio-security.Madin-Darby Canine Kidney (MDCK) and A549  cells were cultured in DMEM  supplemented with 10% FBS . The wild A/Vietnam/1194/2004 (VN1194-W) virus had been propagated two times (35°C) in 10-day-old SPF chick embryos. The serial-propagated A/Vietnam/1194/2004 virus was developed from VN1194-W after four times of proliferations (35°C) in SPF chick embryos and three times of proliferations in MDCK cells (35°C) (VN1194-P). The mouse lung variant (1194-ML) and the mouse brain variant (1194-MB) were isolated separately from the lungs and brains of mice infected with VN1194-P and then propagated for one time in MDCK cells (35°C). The virus of large-plaque variant (1194-LP) and the small-plaque variant (1194-SP) that were isolated from VN1194-P 3 plaque-forming units (pfu) of VN1194-P intranasally (i.n.) and were euthanized on days three post inoculation (p.i.), then their lungs and brains were collected and titrated separately for virus plaque forming assay and virulence determination.Confluent monolayer MDCK cells were inoculated with 10-fold serially diluted H5N1 virus at 35°C for one hour (h). After the removal of the inoculum, cells were washed once with Earle solution and overlaid with 1% hypo-Tm(temperature)-solved agarose containing 0.5% lactalbumin hydrolyzate, 0.5‰ glutamine, and 10% FBS. After two days (d) inoculation at 35°C, cells were stained with 0.25‰ neutral red containing 1% agarose, and the plaque morphology was evaluated. 1194-LP and 1194-SP were serially plaque-purified for three times from the larger plaque and the smaller plaque formed by VN1194-P until homogeneous large or small plaques were formed. Mice were infected with 10MDCK and A549 cells were inoculated with H5N1 virus at a 0.01 MOI (multiplicity of infection), respectively. Then they were maintained in DMEM medium containing 2% FBS at 35°C. At time points of eight, 16, 48 and 72 hours, supernatants of A549 cells, and at time points of eight, 24, 36 and 48 hours, supernatants of MDCK cells were collected and determined by the plaque titer on MDCK cells. The growth curves were determined in three independent experiments.50. The 50% mouse lethal dose (MLD50) of each virus was determined by inoculating five mice in each group with 10-fold serial dilutions of the virus in a 50 μl volume, and calculated by the method of Reed and Muench [50. The remaining 10 mice in each group were monitored for 14 days for mortality and the average survival times of each group, which were also tested by Kaplan-Meier method and Fisher's exact test.Six groups of six to 8-week-old, 15-17 g female BALB/c mice  were lightly anesthetized with pentobarbital and inoculated i.n. with 50 μl (100 pfu) of VN1194-W, VN1194-P, 1194-SP, 1194-LP, 1194-ML, or 1194-MB. 10 mice in each group were monitored for 14 days and the mean survival time was estimated by the Kaplan-Meier method. Fisher's exact test was used to analyze the differences in survival rates when there were no censored observations present. Three mice in groups of VN1194-W, VN1194-P, 1194-LP were euthanized on days three and six p.i.. Mice brains, bloods and lungs were collected and titrated for virus infectivity by TCIDd Muench . In anotVN1194-W, 1194-SP, 1194-LP, 1194-ML and 1194-MB were fully sequenced to find the mutations. Viral RNA was extracted  from virus stocks and reverse-transcribed by reverse transcriptase AMV . Genes of eight influenza fragments were amplified by PCR with a set of fragment specific primers  and DNA polymerase . The PCR products were gel-purified with a QIAquick gel extraction kit  and cloned into pGEM-T-Easy . The nucleotide sequences of the viral genes were analyzed by MEGA 4.The plaques formed by VN1194-W, propagated for only two times in chicken embryo, were homogeneously punctiform in size Figure . The plain vitro passages, the growth of 1194-LP and 1194-SP were determined in MDCK and A549 cells, two commonly used mammalian cells, to confirm whether larger plaques in VN1194-P had been enriched because of higher replication efficiency. Figure Because the larger plaque variants in VN1194-P developed from VN1194-W after serial P < 0.001). This virulence difference was further confirmed by their MLD50 and the mouse average survival time (Table P < 0.001). The weak virulence of 1194-LP was also confirmed by its MLD50 and the mouse average survival time (Table P < 0.001).The virulence of VN1194 with different passage histories was determined in mice. As shown in Figure 50. The positive rate and virus titers in mice brain of VN1194-P group were both lower than VN1194-W group. Same results got by intravenous inoculation, H5N1 virus infected mice efficiently by intravenous inoculation, and VN1194-W and VN1194-P could replicate in the lung, blood and brains, but no virus was detected by TCID50 in the brains of mice infected with 1194-LP.We investigated the correlation between growth characteristics in mice and virulence in mice of those viruses Figure . BecauseP < 0.001) but was less pathogenic than 1194-SP and VN1194-W (Table P < 0.05)To further confirm whether the less neurotropism of 1194-LP isolated from VN1194-P contributed to the attenuation of VN1194-P, we separately isolated the viruses from the lung (1194-ML) and the brain (1194-MB) of mice infected with VN1194-P, and then we investigated the plaque forming property and virulence of two isolates. Similar to VN1194-P, 1194-ML also formed heterogeneous plaques, while 1194-MB only formed smaller plaques whose size was as small as those formed by VN1194-W Figure , Table 160 → Asp in PB2 and another at Met86 → Ile in PA. Same substitutions were also discovered in all other variants in this study. Therefore, 1194-SP is the dominant variant of VN1194-P, inheriting all the genome sequences from VN1194-W. However, 1194-LP fragments had a few substitutions that would contribute to the virus' attenuation. We also found that both 1194-LP and 1194-ML contained three amino acid substitutions in PB2 , two substitution in PB1 , and two substitutions in PA (Ile30 →Thr and Phe46 →Ser). All or part of these homologous mutations contributed to the attenuation and plaque forming property of VN1194-P and 1194-LP. In addition, 1194-LP had more substitutions than 1194-ML, including two in PB1 , two in PA (Lys102 →Arg and Leu106 →Pro) and four in NP .To understand the genetic basis of distinct plaque forming property and virulence of these viruses, we performed sequence analysis for the genomes of VN1194-W and other variants. As in Table Plaque assay is one of the most important procedures for the isolation and titration of viruses like influenza A that forms plaques on MDCK and other cell lines ,28. In tin vitro passages, and only a very low virus titer was determined in one of the three brains of mice after i.n. infection with the VN1194-P. Furthermore, the less virulence of 1194-LP, the larger-plaque variant from VN1194-P, was also associated with no neurotropism in mice. The isolate from the brains (1194-MB) of mice infected with VN1194-P only formed smaller plaques with high virulence in mice. Therefore, the less neurotropism of 1194-LP isolated from VN1194-P may have contributed to the attenuation of the virus.We also discovered that VN1194-W replicated in mouse brain and caused lethal infection after being introduced to the animals intranasally. And we initially observed that this virus' virulence was attenuated in mice after a few th amino acid of PB2 [th amino acid of PB1-F2 [nd amino acid of NS1 [26 → Asn, Ile63 → Thr, and Lys189 → Arg) in PB2, two substitution  in PB1 and two substitutions (Ile30 → Thr and Phe46 →Ser) in PA, all or part of which may cause the attenuation of this virus in mice. In addition, other mutations that only happened in 1194-LP, including two in PB1, two in PA and four in NP, may also be associated with the attenuation in neurotropism in mice.We are interested in understanding how the virulence attenuation of 1194-LP is related to the changes in the viral proteins. Multiple residues at cleavage site of HA ,35, the d of PB2 , the 66tf PB1-F2 , and thed of NS1  have beein vitro culturing. Therefore, the viruses after serial proliferations in vitro may not represent the original viruses in patients or other hosts, emphasizing the importance of direct analysis of original virus samples. Further study on mutations in those proteins may explain the mechanisms of the virulence or neurotropism of H5N1 influenza virus in mice.Our present study demonstrates that less virulent variants are selected by serial passages of VN1194 in embryonated eggs and MDCK cells. The virulence changes were correlated with specific amino acid changes in PB2, PB1, PA and NP proteins, which may have occurred during The authors declare that they have no competing interests.50 and mice survival rate and survival time of viruses. YH, YKX and YQL mainly determined the virus titers in mice tissues and virus growth curve. JL and GHC finished the genetic Sequencing of viruses. JL GHC, YHY and QYZ wrote the manuscript. All authors read and approved the final manuscript.JL, BHL YH, and DWZ carried out plaque forming assay, MLD


Epichloë festucae strains isolated from Festuca rubra plants. In addition, we examined the incidence of infections by E. festucae in populations of F. rubra collected in natural grasslands of Western Spain.In this work we propose a rapid method based on visible and near-infrared (Vis-NIR) spectroscopy to determine the occurrence of double-stranded RNA (dsRNA) viruses in E. festucae isolates were recorded directly from ground and freeze-dried mycelium. To estimate how well the spectra for uninfected and infected fungal samples could be differentiated, we used partial least-squares discriminant analysis (PLS1-DA) and several data pre-treatments to develop calibration models.Vis-NIR spectra (400-2498 nm) from 124 virus-infected and virus-free Applying the best regression model, obtained with two sampling years and using standard normal variate (SNV) combined with first derivative transformation to a new validating data set (42 samples), we obtained a correct classification for 75% of the uninfected isolates and up to 86% of the infected isolates.The results obtained suggest that Vis-NIR spectroscopy is a promising technology for detection of viral infections in fungal samples when an alternative faster approach is desirable. It provides a tool adequately exact and more time- and cost-saving than the conventional reference analysis. Festuca rubra is very common in semiarid grasslands of Western Spain, and wild populations of this species are often infected by the endophytic fungus Epichloë festucae [F. rubra infected by E. festucae, most plants are asymptomatic and produce infected seeds, although a few plants may develop fungal stromata which block the emergence of flowering stems [Epichloë festucae is considered a mutualistic fungus; F. rubra plants infected by E. festucae are more resistant to several species of insect herbivores, because they contain several types of alkaloids produced by the fungus, and have a better appearance and survival rates under stressful conditions than uninfected plants [F. rubra cultivars with Epichloë endophytes is an objective of some turfgrass breeding programs [The perennial grass festucae ,2. In pong stems . Epichlod plants . As a coprograms .Agaricus bisporus [Whereas viruses of plants have long been recognized as important components of plant biosystems, viruses of fungi have been largely ignored. The associations between fungal viruses and their hosts are similar to plant-endophyte associations, because many known fungal viruses cause no obvious symptoms ,6. Only bisporus .E. festucae. EfV1 is a member of the genus Victorivirus (Fam. Totiviridae), and EfV2 is thought to be a member of the Narnaviridae family. Both viruses are efficiently transmitted to asexual spores produced by infected isolates of the fungal endophyte. However, no obvious phenotype is observed in virus infected isolates of E. festucae [The presence of two viruses with genomes of 5.2 kbp (EfV1) and 3.2 kbp (EfV2) has been described in festucae ,9.Although several methods, including enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), immunofluorescent assay and Western blotting have been used for the diagnosis of viral infections, none of them is ideal in terms of cost-effectiveness, speed, and accuracy. Currently, the rate of outbreak of emerging viruses is increasing and therefore the development and establishment of analytical methods for such viral infections are becoming more important .Near-infrared (NIR) spectroscopy is a fast, accurate and non-destructive technique that does not require chemical reagents. Visible and NIR spectroscopy is the method which covers the region from 400 to 2500 nm. The absorption of molecules in the NIR region is due to combinations and overtones of vibration such as stretching and bending of hydrogen-bearing functional groups like -CH, -OH, and -NH . The devNowadays, NIR spectroscopy has been applied successfully in many fields such as agriculture, environment, and medicine, as well as in the pharmaceutical, chemical, petrochemical and food industries . HoweverE. festucae. This technique could be useful as a quick identification tool to give solid clues before confirmation by a genotypic method. In addition, a field study was done to determine the frequency of infection by the endophyte E. festucae in F. rubra plants, and the incidence of viruses in these populations of E. festucae.The objective of this study was to develop models using spectral Vis-NIR reflectance measurements to discriminate between virus infected and virus free strains of the endophytic fungus E. festucae were obtained during the spring of 2008 from 161 plants of F. rubra collected at six different locations in natural semiarid grasslands of the province of Salamanca, in Western Spain. At each location between 21 and 34 plants were dug out, leaving a distance of at least 10 m from each other, and transported to the laboratory. Epichloë festucae was isolated from plant stems and leaf sheaths surface disinfected with a 15 minute treatment with a solution of 20% commercial bleach (1% active chlorine). After rinsing in sterile water, about 15 stem pieces were placed on each of 2 Petri plates containing potato dextrose agar (PDA) with 200 mg/l of chloramphenicol and incubated at 24°C in the dark. It takes about one week for E. festucae mycelium to emerge from plant fragments [E. festucae on the basis of morphological characteristics of the cultures [Isolates of ragments ; therefocultures .To produce enough mycelium for dsRNA extraction and NIR analyses, each isolate was grown on top of sterile cellophane disks layered on top of PDA plates. The mycelium obtained from eight plates was freeze-dried and ground in an electric coffee grinder.E. festucae isolates obtained from two locations  in the year 2006 were used. These isolates were also grown on top of sterile cellophane disks in eight PDA plates, and their mycelium was freeze-dried, but the grinding was done with liquid nitrogen in a mortar. These samples were included in the NIR analysis to add more universality, and to account for more variability , but were not considered for the estimation of endophytic and virus infection frequencies.In addition, for the NIR study 31 E. festucae from 2008, and 31 from 2006 by means of the detection of the 5.1 and 3.2 kbp dsRNA molecules which correspond to the genomes or replicative intermediates of the viruses. The dsRNA was purified from 0.5 - 1 g of mycelium by CF-11 cellulose chromatography [The presence of the viruses EfV1 and EfV2 was diagnosed using 93 isolates of For the acquisition of spectra, five freeze-dried and ground cultures of each isolate were scanned on a NIRSystem 6500 scanning monochromator  fitted with a sample transport module. The pulverized mycelium was placed on a circular sample cell with a quartz window (38 mm in diameter and 10 mm in thickness). ISIscan version 3.10 and WinISI 4 software  were used to control and diagnose instrument performance and to manage the recorded spectra. Spectra from 400 to 2500 nm were acquired at 2 nm wavelength increments, obtaining 1050 data points for each spectrum. Visible and near infrared measurements were made in reflectance mode (R). The average of the five spectra obtained from each isolate was used in the following analyses. A total of 124 mean spectra were recorded as log (1/R), where R is the intensity of reflected light at each wavelength. The laboratory temperature was kept at 24°C during NIR measurements.For data analysis, spectral (log 1/R) files were exported from WinISI in NSAS format to The Unscrambler software . Calibrations generated for the classification of virus infected and virus free isolates were developed and evaluated with separated calibration and prediction sample sets. The calibration set consisted of two-thirds of the samples , and the validation set consisted of the remaining third . Samples were assigned to each set based on their position in the spectral file; thus, every third sample was used for external validation and the remainder for calibration.a priori knowledge about the category membership of samples, was used in the qualitative analysis. Partial least-squares discriminant analysis (PLS-DA) was the method used to generate calibrations for the classification. It is a method that compresses the spectral data into orthogonal structures called factors, which describes the maximum covariance between the spectral information and the reference values. Successful compression using only a few factors is often possible in spectroscopy because the numerous data points are highly co-linear [Previous to the development of the classification models, principal component analysis (PCA) was made using The Unscrambler software to inspect spectral patterns for improved understanding, and to detect outliers or any clustering of the data . A supero-linear ,22.Because categorical predictor variables cannot be entered directly into a regression model and be meaningfully interpreted, some other method of dealing with information of this type must be developed. In this paper, PLS1-DA onto a dummy variable  was usedPre-treatments of the spectral data are often applied in an effort to improve calibration accuracy. In order to obtain the best discrimination model, nine different types of spectral pre-treatments were tested. These included no treatment , multiplicative scatter correction (MSC), standard normal variate (SNV), first derivative (1D), second derivative (2D), and MSC or SNV followed by derivative transformations (Savistsky Golay method). Particular derivative parameters used were 1D  and 2D , in which the first and second numbers are the left and right number of side points, and the third is the polynomial order. A more detailed description of these data pre-treatments can be found in Petisco et al. .E. festucae. The infection frequencies ranged from 24% to 87%  according to infection for the external validation sets are shown in Table E. festucae isolates (infected and uninfected) when applied the data transformed to 2D or its combination with MSC or SNV  and 1950 nm (O-H combination bands) Figure . Other aPrior to discriminant analysis, principal component analysis (PCA) was used to obtain preliminary indications of the possible clustering of the samples in the two classes: fungal isolates infected and uninfected by viruses. We found two sample clusters in the PCA and PLS1 scores plots Figures  and 4 coThe differences in classification between years could be due to the different ways used for grinding the fungal samples each year, since the particle size affects strongly to the absorbance of the spectra. Also, culture age was different for the 2006 and 2008 samples. Therefore, for best results it seems very important to process all the samples exactly in the same way, using cultures of the same age and pulverizing the freeze-dried mycelium in liquid nitrogen. In addition, the better separation of virus infected and virus free samples in 2006 could be due to the fact that among the 93 samples of 2008, infections by EfV2, as well as mixed infections by both viruses occurred. In contrast, all the virus infected samples from 2006 were infected by the two viruses, being their virus load higher. Therefore the best results of 2006 could also be related to a lower variability in samples.Epichloë virus genomes are composed of dsRNA, and high molecular weight dsRNA is an specific virus-related compound which is absent from virus-free plants and fungi [E. festucae strains is high, particularly in those infected by EfV1 [Epichloë can be discriminated.nd fungi . The dsR by EfV1 . TherefoExperience with NIR data has shown that PLS-DA can give good prediction results in the classification of similar fungal species ,27 or seE. festucae. This technique might be useful for other situations were fungi are infected by viruses, for example for the diagnosis of La France disease in mushrooms. Currently the detection of fungal viruses is based on techniques like CF11 chromatography, which are a laborious conventional method of analysis.This study is the first to demonstrate that Vis-NIR spectroscopy combined with PLS1-DA method is able to detect viral infections in a fungus, E. festucae in F. rubra plants and in E. festucae isolates by viruses. The application of the PLS-DA algorithm to visible and near-infrared spectra of fungal isolates was found to be a promising method for the rapid detection of virus infection.. In all cases, pre-processing was advantageous to improve interpretability and classification ability.The results of this work suggest relatively high levels of infection by Further work will be necessary to standardize sample presentation and spectral collection and to optimize the calibration procedures for validating the obtained calibration models with independent samples from other locations or years. Anyway, although the results obtained in this study could be considered as preliminary, the technique may be considered as promising for future studies including large number of samples infected with other viruses associated to plant pathogenic fungi.The authors declare that they have no competing interests.CP carried out the experiments and drafted the manuscript. AGC and IZ supervised all experiments and provided discussion for the preparation of the final report. IZ and BGC participated in sample collection and BRVA participated in the revision of the manuscript. All authors have read and approved the final manuscript.


Miniopterus schreibersii) in Spain. It does not belong to phylogroups I or II, and it seems to be more closely related to the West Causasian bat virus, and especially to the Ikoma lyssavirus.A new tentative lyssavirus, Lleida bat lyssavirus, was found in a bent-winged bat ( Lyssavirus, 12 species have been described: Rabies virus (RABV), Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssavirus types 1 and 2 (EBLV-1 and -2), Australian bat lyssavirus (ABLV), Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV), West Causasian bat virus (WCBV), and Shimoni bat virus (SHIBV). Two more recently described viruses have not yet been classified: Bokeloh bat lyssavirus (BBLV)  and phylogroup II . Phylogroup III consists of WCBV . The bat arrived lethargic and dehydrated, died soon after admission, and its carcass was frozen at −20°C. On March 12, 2012, as part of the rabies surveillance program in Spain, the bat carcass was received by the National Center of Microbiology, where rabies testing was conducted by 2 generic reverse transcription PCR (RT-PCR) methods for lyssavirus detection  and genetically identified by cytochrome b sequencing (http://blast.ncbi.nlm.nih.gov/), showed no substantial sequence similarity to previously known lyssaviruses.The bat was morphologically identified as a bent-winged bat (http://mrbayes.csit.fsu.edu/). Two simultaneous runs of 106 generations were conducted, each with 4 Markov chains, and trees were sampled every 100 generations. The best-fit nucleotide model, GTR + I + G, available in MrBayes was selected according to the corrected Akaike information criterion. The phylogenetic reconstruction was based on a dataset representative of all known lyssaviruses, including the recently described IKOV. The topology obtained showed that this sequence is more closely related to IKOV and WCBV than to the lyssaviruses in phylogroups I and II .To determine the identity of the lyssavirus, we sequenced a larger fragment (565 bp), including the variable codifying region of the nucleoprotein gene (GenBank accession number submitted). We reconstructed an overall phylogeny of lyssaviruses by using a Bayesian Inference with the first 405 nt of the N-gene and MrBayes version 3.1.2 (I and II . These rThe highest nucleotide identity was with IKOV (71.6%), followed by SHIBV (68.6%), IRKV (68.1%), KHUV (67.6%), EBLV-2 (67%–68.2%), ARAV (67.3%), WCBV (67.4%), ABLV (66.6%–67.7%), BBLV (66.1%), LBV (65.7%–68.6%), MOKV (65.7%–67.2%), DUVV (65.5%–65.8%), RABV (64.7–66.4%), and EBLV-1 (63.7%–64%). The lowest nucleotide identity was with the only lyssavirus found in bats of the Iberian Peninsula, EBLV-1. The nucleotide identity among the previously known lyssaviruses was 63.5%–80.0% in this particular fragment, and the lowest identities among strains belonging to the same lyssavirus were 80.4% for ABLV and 79.9% for LBV (the most distant LBV strain has been suggested to be a different lyssavirus) .The lyssavirus-specific antigen reactivity and association with a genomic sequence found in a bent-winged bat in northeastern Spain could be derived from the tentative new virus LLEBV. According to our phylogenetic reconstruction, the virus does not seem to belong to phylogroup I, which comprises most bat lyssaviruses, or to the African phylogroup II. The evolutionary relationships between the LLEBV sequence with WCBV and IKOV sequences need to be clarified before it can be determined whether they form >1 different phylogroups. M. schreibersii bats, as was WCBV, the other European lyssavirus outside phylogroup I. The genus Miniopterus has traditionally been considered to belong to the family Vespertilionidae as do other bat genera linked to lyssaviruses in Eurasia . However, recent molecular analyses have confirmed that the genus Miniopterus belongs to the family Miniopteridae  of these bats overwinter in caves and move in the spring to different and sometimes distant summer roosts for reproduction  and similarities . Analysis was performed with the 405-nt fragment of the N-gene of all the known


Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16− and CD16+ blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16+ monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16+ monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease.Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16 CD16− monocytes are predisposed to produce IL-10, while CD16+ monocytes produce more pro-inflammatory cytokines, for example TNF-α − express CCR2 while CD16+ monocytes preferentially express CX3CR1 + monocytes are elevated during several inflammatory and infectious conditions, and have been proposed to play pro-inflammatory roles during diseases + monocyte subset has also been shown to be more permissive to HIV infection The human peripheral blood monocyte population can be divided into two distinct subsets based on CD16 expression Flaviviridae. Dengue virus exists as four serotypes, DENV-1 to 4 and is transmitted to humans via the bite of an Aedes mosquito Dengue virus is a single stranded positive sense RNA virus that belongs to the family + activated monocytes were found in dengue patients Monocytes are natural host cells for dengue virus In vitro, pre-treatment of human primary monocytes with Th2 cytokines, including IL-4 and IL-13, results in enhanced susceptibility to dengue virus Evidence suggests that severe manifestations of dengue could be caused by an inappropriate Th2-biased immune response. For example, levels of IL-13 are elevated in patients with dengue hemorrhagic fever + and CD16− human blood monocyte subsets from healthy donors and exposed them to dengue virus (DENV2 NGC) in vitro. Our results revealed that both monocyte subsets are similarly susceptible to dengue virus infection. The two monocyte subsets were also able to effectively produce protective anti-viral soluble factors. However, monocyte subsets differed in the production of inflammatory soluble factors associated with dengue pathogenesis and inflammation. Under Th2 conditions mimicked by IL-4 treatment, the susceptibility of monocyte subsets was enhanced, but this affected the CD16+ monocyte more than the CD16− monocyte subset. The results herein reveal that monocyte subsets can have similar susceptibility and protective mechanisms against dengue, but may fundamentally differ in the mechanisms of dengue pathogenesis.Given the vital role of monocytes in dengue virus infection, it is important to delineate how monocyte subsets could differently support dengue virus replication and contribute to the protective and pathogenic manifestations of dengue. In order to investigate the differential roles of monocyte subsets in a primary dengue virus infection setting, we isolated the CD16− and CD16+ monocyte subsets were isolated using the CD16 monocyte isolation kit  according to the manufacturer’s instructions, with some modifications. Briefly, NK cells, neutrophils and T cells were depleted using microbeads conjugated to anti-CD56, anti-CD15 and anti-CD3 antibodies. After depletion, CD16+ monocytes were isolated using anti-CD16 conjugated microbeads. CD16− monocytes were subsequently isolated from the negative fraction using anti-CD14 conjugated microbeads. The purity of the isolated monocyte subsets was consistently >90%.All blood samples and procedures in this study were approved by the National University of Singapore Institutional Review Board, approval number NUS 1076, in accordance to guidelines of the Health Sciences Authority of Singapore. Informed written consent was taken by the staff of the blood bank of the Health Science Authority of Singapore, in accordance to the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were obtained from blood of healthy donors using Ficoll-Hypaque  density gradient centrifugation at 1200×g for 30 mins at room temperature. CD165 cells per well in 200 µl of RPMI 1640 medium  supplemented with 100 µg/100 U/ml Streptomycin/Penicillin (Sigma-Aldrich) and 10% Fetal bovine serum (FBS) (Gibco). All cells were cultured in a 37°C 5% CO2 incubator. For protection assays, K562 cells  were pre-treated for 24 h with supernatants from monocyte subsets with or without dengue virus exposure. Prior to pre-treatment, these supernatants were filtered through 100 kDa centrifuge filters  at 3000×g, 45 mins at room temperature, to remove virus by size exclusion. Pre-treated K562 cells were washed and infected with DENV2 NGC at a MOI of 2. After 1 h, cells were washed and cultured in RPMI 1640 media with 10% FBS. After 2 days, the extent of infection was determined by flow cytometry using anti-NS1  intracellular labeling. Data were collected on a BD FACS calibur .Following isolation, monocytes were exposed to DENV2 NGC at a multiplicity of infection (MOI) of 10. After 3 h, monocyte subsets were washed twice and cultured in 96 well U-bottomed plates at 2×10DENV 2 NGC was used for this study. For virus propagation, C6/36 cells were initially exposed to the virus at MOI of 0.01. After 1 h, the cells were washed and cultured at 28°C in RPMI 1640 supplemented with 5% FBS. At days 4, 5 and 6, cell supernatants were harvested. Cell debris was removed by centrifugation at 1000×g for 10 mins and stored at −80°C. Virus titers were determined by plaque assay.) cells were seeded into 24 well plates and grown to confluence. Serially diluted supernatants containing dengue virus were added to the cells and incubated for 1 h at 37°C in a 5% CO2 incubator. Cells were then overlaid with RPMI medium containing 0.4% carboxymethylcellulose  and incubated for 4 1/2 days at 37°C in a 5% CO2 incubator. The cells were then fixed with 1% formalin before plaques were revealed by staining with 1% crystal violet . Virus titres are thus expressed as plaque forming units per milliliter (PFU/ml).BHK-21   according to the manufacturer’s instructions. Plates were read using a Luminex 200 . IFN-α levels were determined using IFN-α multi-subtype ELISA kit according to the manufacturer’s instructions . Absorbance at OD450 nm was measured by a Tecan M200 microplate reader .Cells were fixed with PBS containing 4% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature, before permeabilization with PBS containing 0.5% BSA and 0.1% saponin (Sigma-Aldrich). Cells were labeled with anti-NS1 PE  and anti-E-protein (Envelope protein) (4G2) allophycocyanin  for 45 mins at room temperature. Following washing with permeabilization buffer the cells were analyzed using a FACS Calibur (BD Bioscience).2 and 10 mM Hepes/NaOH. Cells were subsequently stained with Annexin-V PE (BD Bioscience) and 7-AAD  for 15 min and analysed by flow cytometry. Live and dead cells were also discriminated using a Live/Dead® cell stain kit (Invitrogen) comprising a far red reactive dye with an emission of 660 nm. Live cells actively exclude this dye and stain dimly, while dead cells are unable to exclude the dye and stain brightly. Staining was performed in PBS according to the manufacturer’s protocol, before analysis by flow cytometry. A MTS assay which detects the dehydrogenase activity of metabolically active cells was performed using CellTiter 96® AQueous kit  according to the manufacturer’s protocol. Dehydrogenase activity converts MTS into a formazan product was measured by its absorbance at OD490 nm using a Tecan M200 microplate reader. Absorbance at OD490 nm is directly proportional to the number of viable cells in culture.For Annexin V and 7-AAD (7-Aminoactinomycin D) staining, cells were washed and resuspended in Annexin V buffer containing 140 nM NaCl, 2.5 mM CaCl6 cells/ml. Monocyte subsets were treated with recombinant human IL-4 (Immunotools) at concentration of 25 ng/ml. As controls, monocyte subsets were cultured without IL-4 treatment. The cells were incubated at 37°C, 5% CO2 for 2 days. The monocyte subsets were washed with culture media prior to exposure to dengue virus at a MOI of 10.Isolated monocyte subsets were seeded into flat-bottom 6-well plates at a cell density of 1×10t test. Differences with p<0.05 were considered significant.The statistical significance of the results was determined by a two-tailed Student’s − and CD16+ monocyte subsets to dengue virus infection. CD16− and CD16+ monocytes were isolated from PBMCs of healthy blood donors. Typical purities of the isolated monocyte subsets are shown in + and CD16− monocytes after virus exposure. These results show that both monocyte subsets were equally susceptible to dengue virus infection.We first compared the susceptibility of CD16We next determined whether both monocyte subsets had the ability to support production of infectious virus particles. The supernatants of virus exposed monocyte subsets were harvested over the course of six days and tested for the presence of newly produced infective virus by plaque assay on BHK-21 cells . At day + and CD16− virus exposed monocytes. Hence, both monocyte subsets were equally capable of producing protective factors against dengue virus, although the major part of this response occurred late (>4 days) after exposure to virus.Cytokines and chemokines produced by monocytes play vital roles in dengue pathogenesis and protection 5 fold, but retains the levels of soluble factors by >80% (data not shown). For infection, we used K562 cells which are highly susceptible to infection by dengue virus − and CD16+ monocytes, no significant difference in the reduction of K562 infection was observed. Hence, monocyte subsets infected with dengue virus produced protective factors, including IFN-alpha and IP-10. Hence, we speculate that these protective factors present in supernatants used to treat K562 cells protected them from infection by dengue virus.Next, we determined the ability of virus exposed monocyte subsets to confer protection against dengue virus infection. Supernatants of monocyte subsets 6 days after infection were used, since they contained the highest levels of factors associated with protection against dengue. Residual virus in these supernatants was first removed by size exclusion using 100 kDa centrifuge filters. This efficiently reduces infective virus loads by 10+ monocytes were significantly higher than that of CD16− monocytes in response to dengue virus. Virus exposed CD16+ monocytes produced picogram levels of IL-1β and TNF-α, but these cytokines were undetectable with CD16− monocytes (+ monocytes was more than 30 times higher than the levels observed with CD16− monocytes at day 2 (1939.9±717.2 versus 58.1±21.0)  . Hence, + and CD16− monocytes. The levels of CCL2, 3 and 4 were significantly higher in supernatants of virus exposed CD16+ monocytes compared to virus exposed CD16− monocytes. The level of CCL2 was more than five fold higher for virus exposed CD16+ versus CD16− monocytes at any time tested  , CCL3 (MIP-1α) and CCL4 (MIP-1β), was also clearly different between CD16e tested . While tcultures . Similar570±195) . The pro− and CD16+ monocytes. To determine whether the viability of monocyte subsets was indeed increased upon virus exposure, we stained the cells with Annexin V and 7-AAD over the course of 6 days (-7-AAD-) for both monocyte subsets following exposure to virus compared to monocytes without virus. - 7-AAD- cells over the course of 6 days. We also used a Live/Dead cell stain kit that consists of a dye that emits at 660 nm to determine viability. Live cells actively exclude this dye and stain dimly, while dead cells are unable to exclude the dye and stain brightly. The percentages of viable (dimly stained) and dead cells (brightly stained) in virus exposed or monocyte subsets without virus were determined by flow cytometry  populations reveal that positive selection did not affect cellular transcription in any significant manner There might also be concerns on whether the results obtained from the lab adapted NGC strain is best suited for infecting primary cells. However, other groups have also successfully infected human monocytes using the 16681 strain, and have found comparable results between the 16681 and NGC strain + monocyte subset, the CD14high intermediate subset was selectively expanded in mild but not severe dengue Overall, our results delineate how monocyte subsets can have both similar and different responses to dengue virus infection. This demonstrates the advantage of taking monocyte heterogeneity into account for studies of viral infections like dengue. More recently, monocytes have been further subdivided into three subsets Figure S1Infection of whole PBMCs. Whole 1 PBMCs were were exposed to dengue virus or medium without virus. After two days, surface staining of CD14 and CD16, and intracellular staining for NS1 and 4G2 was performed. (A) NS1 and 4G2 expression by gating on the whole CD14+ monocyte population. (B) NS1 and 4G2 expression by gating on the whole CD14− non monocyte population. (C) CD14 and CD16 profiles demonstrating the spontaneous upregulation of CD16 by monocytes after two days of culture. (D) NS1 and 4G2 expression by gating on the CD14+CD16+ monocyte population (E) NS1 and 4G2 expression by gating on the CD14+CD16 monocyte population. (F) Percentage of NS1+ and 4G2 positive cells from the mean + SD of three donors.(PDF)Click here for additional data file.


Viral encephalomyelitis is an important cause of morbidity and mortality worldwide, and many encephalitic viruses are emerging and re-emerging due to changes in virulence, spread to new geographic regions, and adaptation to new hosts and vectors The primary target cells for most encephalitic viruses are neurons, although a few viruses attack cerebrovascular endothelial cells to cause ischemia and stroke or glial cells to cause demyelination, encephalopathy, or dementia Because infections with encephalitic viruses are initiated outside the CNS , innate and adaptive immune responses are usually mounted rapidly enough to prevent virus entry into the CNS. Therefore, most viruses that can cause encephalitis more often cause asymptomatic infection or a febrile illness without neurologic disease, and encephalomyelitis is an uncommon complication of infection.When a virus does invade the CNS, there are several routes by which infection of neurons can occur. The most common entry point is from the blood, and the level of viremia as a result of virus replication in peripheral organs often correlates with the likelihood of CNS infection. However, the blood–brain barrier (BBB), composed of vascular endothelial cells with tight junctions in contact with the foot processes of astrocytes, inhibits direct access to the brain parenchyma and neurons. Some neurotropic viruses can replicate in cerebrovascular endothelial cells, enter with infected leukocytes, or cross directly into the cerebrospinal fluid (CSF) through the porous capillaries of the choroid plexus. A specialized CNS entry pathway used by several viruses, most notably HSV, varicella zoster, and rabies viruses, is by way of nerve terminals in peripheral organs. These viruses can enter the nerve and then use neural transport mechanisms to transport the infecting virions to the neuronal cell body where replication occurs In tissue culture systems most of these viruses can infect many types of cells, in addition to neurons. Few neuron-specific virus receptors have been identified  but these do not always account for neuronotropism in vivo In addition to fever and headache, signs and symptoms of viral encephalomyelitis typically include evidence of neuronal dysfunction—seizures, cognitive impairment, ataxia, paralysis, etc. Virus replication can damage neurons directly by inducing cell death through apoptotic or necrotic mechanisms  [14]. MaAlthough neuronal virus infection per se is necessary for, and contributes directly to, neuronal dysfunction, the inflammatory response in the CNS is also a major contributor to neuronal damage and can even result in death of nearby uninfected neurons  [19], [2+ T cells) or of MHC class II (CD4+ T cells) and can possess cytotoxic properties. Neurons are long-lived essential cells that cannot be replaced, so a noncytolytic immune mechanism for virus clearance would be advantageous to the host to avoid death of surviving cells. If the immune clearance mechanism is damaging to the infected neuron, then the function of that neuron will be lost and the outcome for the host will be the same as if the virus infection had caused neuronal death.The immune response to infection can contribute to fatal disease, but is also necessary for recovery and virus clearance. Elimination of virus-infected cells from tissue requires elimination of all cells in which the virus is replicating. This can occur either by virus-induced or immune-mediated cytolysis. T cells are ideal for this purpose because they recognize viral antigen as processed antigen only in the context of cell surface–expressed MHC class I (CD8Because mature neurons are relatively resistant to virus-induced cell death Because the noncytolytic process for virus clearance does not completely eliminate viral RNA from neurons, a mechanism for long-term immunologic control of virus replication is needed to prevent virus reactivation or progressive disease Encephalomyelitis resulting from virus infection of neurons is a disease that can be fatal or result in permanent disability due to irreversible damage of infected neurons. The immune response to infection can enhance neuronal damage or can control virus replication by noncytolytic mechanisms and thus determine outcome. However, noncytolytic virus clearance results in persistence of viral nucleic acid in the CNS and thus establishes a need for long-term local immune responses to prevent reactivation of infection and progressive disease. Understanding these mechanisms is necessary for development of strategies for treating and preventing neurologic disease due to viral encephalomyelitis.


Flavivirus, AHFV) was recently identified as the agent of a viral hemorrhagic fever in Saudi Arabia and characterized serologically and genetically as a variant genotype of Kyasanur Forest disease virus (KFDV). Since viral diagnosis and vaccine development may be hindered by genetic diversity, this study was intended to address AHFV genetic heterogeneity. Eleven strains isolated from hospitalized patients from 1994 to 1999 in Saudi Arabia were sequenced in the envelope, NS3, and NS5 genes. Homologous sequences were compared and used to look for patterns reflecting specific evolution associated with spatiality, temporality, infection pathway, and disease prognosis. Genetic analyses showed low diversity, which suggests a slow microevolution. Evaluation of divergence times showed that AHFV and KFDV ancestral lineage diverged 66–177 years ago, and the diversity observed within the studied AHFV strains reflected a 4- to 72-year period of evolution.Alkhurma hemorrhagic fever virus (genus  Alkhurma hemorrhagic fever virus (AHFV) was first isolated in Jeddah, Saudi Arabia, in the 1990s from the blood of a butcher admitted to the hospital with a severe infectious syndrome. To date, 24 cases have been recorded in a 10-year period. Clinical manifestations include fever, headache, retroorbital pain, joint pain, generalized muscle pain, anorexia and vomiting associated with leukopenia, thrombocytopenia, and elevated levels of liver enzymes. In addition, some patients had clinical symptoms of hemorrhagic fever or encephalitis; overall, 5 patients, of 24 infected, died, for a 25% fatality rate  amplification of a 220-bp genome fragment that exhibited 89% nucleotide (nt) sequence homology with the Kyasanur Forest disease virus (KFDV) NS5 gene. Recently, the complete coding sequence of AHFV was determined; comparative analysis with other tickborne flaviviruses confirmed that AHFV was most closely related to KFDV, and genetic distances suggested that AHFV was a subtype of KFDV  and shipped to the Unité des Virus Emergents laboratory in Marseille, France.2, 50 mmol/L KCl, 0.1% Triton X-100, 200 μmol/L each deoxynucleoside triphosphate, 0.2 μmol/L of each primer, 3 μL of cDNA and 1.5 U of Taq DNA polymerase . The thermocycler profile was 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 1 min at 55°C, and 2 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products of the expected size were purified from agarose gel slices with the Wizard PCR Preps DNA Purification System (Promega). Both strands of each PCR product were sequenced directly, with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 on an Applied Biosystems 3730x1 DNA Analyzer  at the company Genome Express .RNA was purified in a BSL-3 laboratory according to the manufacturer's instructions. Reverse transcription (RT) of virus-specific RNA was carried out at 42°C in a 20-μL reaction that included 11 μL of RNA extract, 200 U of Superscript IITM RNase H-Reverse Transcriptase , and 1 pmol of primer ALK-NS5R. Truncated noninfectious cDNA molecules were produced. PCR products were generated independently from the envelope, NS3 region, and NS5 region by using ALK-ES/ALK-ER, ALK-NS3S/ALK-NS3R, and ALK-NS5S/ALK-NS5R pairs of primers, respectively. PCR reactions were carried out in a volume of 100 μL that included 10 mmol/L Tris-HCl (pH 9.0), 1.5 mmol/L MgClSequences from the 11 AHFV isolates were compared with homologous sequences representing other tickborne flaviviruses retrieved from the GenBank database: Langat virus (LGTV) M73835), Powassan virus (POWV) L06436), deer tick virus (DTV) (AF311056), Omsk hemorrhagic fever virus (OHFV) (AY323489), Kyasanur Forest disease virus (KFDV) , tick-borne encephalitis virus (TBEV) Neudoerfl strain (U27495), Hypr strain (U39392), Vasilchenko strain (L40361), Sofjin strain (AB062064), and Louping ill virus (LIV) (Y07863). Nonvectored flaviviruses (Rio Bravo virus [NC_003675] and Apoi virus [NC_003676]) were used to root the phylogenetic tree. The nucleotide sequence alignments were generated with Clustal W 1.7 . Distances observed by using nonsynonymous sites were calculated according to the Nei and Gojobori algorithm implemented in MEGA  are involved in viral transmission and that they exhibit different seasonal peaks; similarly, each developmental stage has a peak of activity that corresponds to different seasons. Whether AHFV has similar features is unknown, but if so this finding could account for 2 peaks of cases during the year . Two additional cases reported through ProMED in 2002 and 2004 , as previously demonstrated for other flaviviruses vectored by ticks . Based oAlthough the human cases were acquired from different sources and through different routes, the clinical and biological features were similar; whether asymptomatic infection may occur is not known and would require seroepidemiologic studies. From 1999 to 2004, the ProMED Web site reported on 3 occasions a total of 9 cases of infection; at least 2 were fatal. The overall rate of death observed with AHFV  appears to be much higher than that currently reported for KFDV (3.0%–8.9%) ,15 and sThe genetic variability between the different isolates was low regardless of 1) time of the year during which the strain was recovered, 2) disease symptoms in the infected patients, 3) presumed route of infection, and 4) environment in which the patient lived. This pattern was observed for KFDV isolates in India, although these conclusions were established from antigenic methods rather than sequence analyses . This evEvolution of AHFV and closely related tickborne flaviviruses was addressed on the basis of previous estimation of the times of divergence of flavivirus species  Figure . By usinThe high genetic similarity between all AHFV strains makes designing and developing specific and sensitive RT-PCR diagnostic assays possible. Close antigenic properties are also important for vaccine development. Among the many issues that merit future investigations, cross-protection conferred by KFDV and commercially available tick-borne encephalitis virus vaccines should be tested since slaughterhouse workers appear to rank high on the risk scale and therefore may be the first population to benefit from this information.Seroepidemiologic studies are needed in various population groups to determine the extent of AHFV infection and its geographic distribution in the Middle East peninsula . Studies are needed to achieve a better understanding of the natural history of this virus but its pathogenicity in humans, specifically the prevalence of asymptomatic and symptomatic cases. The most important issues to resolve are the origin of the virus, how it is dispersed, and how it came to be in Saudi Arabia so that disease control strategies can be devised.


The role of relative humidity in the aerosol transmission of influenza was examined in a simulated examination room containing coughing and breathing manikins.Nebulized influenza was coughed into the examination room and Bioaerosol samplers collected size-fractionated aerosols  adjacent to the breathing manikin’s mouth and also at other locations within the room. At constant temperature, the RH was varied from 7–73% and infectivity was assessed by the viral plaque assay.Total virus collected for 60 minutes retained 70.6–77.3% infectivity at relative humidity ≤23% but only 14.6–22.2% at relative humidity ≥43%. Analysis of the individual aerosol fractions showed a similar loss in infectivity among the fractions. Time interval analysis showed that most of the loss in infectivity within each aerosol fraction occurred 0–15 minutes after coughing. Thereafter, losses in infectivity continued up to 5 hours after coughing, however, the rate of decline at 45% relative humidity was not statistically different than that at 20% regardless of the aerosol fraction analyzed.At low relative humidity, influenza retains maximal infectivity and inactivation of the virus at higher relative humidity occurs rapidly after coughing. Although virus carried on aerosol particles <4 µM have the potential for remaining suspended in air currents longer and traveling further distances than those on larger particles, their rapid inactivation at high humidity tempers this concern. Maintaining indoor relative humidity >40% will significantly reduce the infectivity of aerosolized virus. Winter influenza outbreaks occur with seasonal regularity in temperate climates and it has been suggested that humidity may affect transmission During the winter, people spend the majority of their time indoors and the risk of aerosol transmission of influenza by coughing, sneezing and breathing is a concern because respirable particles carrying influenza may remain airborne for prolonged periods. Influenza RNA has been detected in the exhaled breath and coughs of patients with influenza Healthcare workers treating influenza patients are particularly prone to infection as they can be exposed to multiple patients in closed examination rooms over the course of a day. A novel approach to assess risk factors is the use of manikins in a controlled environment. This approach has been used to study the flow of human respired air in a room To address whether humidity contributes to the risk of aerosol transmission of influenza, a simulated examination room equipped with environmental controls was constructed that contained a coughing and breathing manikin to simulate a healthcare worker’s exposure Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) and Influenza strain A/WS/33  were purchased from the American Type Culture Collection  and maintained as described National Institute for Occupational Safety and Health (NIOSH) bioaerosol samplers, which collect and size-fractionate aerosols into three fractions , were used to collect influenza-containing aerosols The amount of total virus (infectious and non-infectious) in an aerosol sample was determined by real-time qPCR analysis to assess the number of Matrix1 gene copies as described The number of infectious virus within an aerosol sample was determined by the VPA. Aerosols containing infectious influenza were inoculated onto a confluent lawn of MDCK cells and plaque forming units (PFU) were calculated as described The simulated examination room (aerosol exposure simulation chamber) is 3.16 m×3.16 m×2.27 m high and includes a HEPA filter and an ultraviolet lamp The analysis of the number of PFUs induced by viral particles collected from the samplers was generated using SAS/STAT software, Version 9.2 of the SAS system for Windows . Data were transformed by calculating the natural log of PFUs prior to analysis to meet the assumptions of the statistical tests (homogeneity of variance). For samples collected for 60 minutes under 7 different RHs, a two-way factorial mixed-model analysis of variance (ANOVA) was performed on RH and fraction. This was done using RH as a numeric independent variable to calculate slopes, as well as a categorical variable to allow comparisons between mean levels of PFUs in each fraction at each RH. A significant interaction in a model with humidity as a numeric variable indicates that the slopes of the lines which plot PFUs as a function of RH are not equal across fractions. The second experiment, which sampled for 15 minute intervals for 60 minutes at 2 different RHs was analyzed with a three-way factorial mixed model ANOVA on RH, time and fraction, each being utilized as class variables. The final experiment which sampled for 60 minutes between hours 4 and 5 following aerosol generation was analyzed using a two-way mixed model ANOVA on RH and fraction. In all analyses, trial was included as a random variable in ‘Proc Mixed’ to account for the lack of independence between fractions in a given trial. Interactions were analyzed by examining simple main effects using the ‘slice’ option. All pairwise comparisons were considered significant at p<0.05.8 total virus was coughed into the exam room which equilibrated to 4.5×103 total virus/per liter of room air (assessed by qPCR Matrix gene copies). A particle counter positioned just below the coughing manikin’s mouth showed that the coughed particles optical diameters were largely within the respirable size range . The percentage of virus that retained infectivity (number of PFUs/number of qPCR Matrix copies in an aerosol sample) relative to that prior to coughing was determined to be highest (70.6–77.2%) at 7–23% RH with a dramatic drop to the lowest (14.6%) at 43% RH  apart . Approxize range . Most oft 43% RH . Increast 43% RH . Specifi6 at 20% RH and 1.4×106 at 45% RH  after coughing and compared at 20% and 45% RH. The total amount of virus (assessed by qPCR of the matrix gene) collected during the initial 60 minutes after coughing was 1.8×10t 45% RH . During t 45% RH . Within t 45% RH . Continut 45% RH .5 virus) and 45% RH (9.9×104 virus) (The amount of infectious virus present in the 3 aerosol fractions was then assessed to determine whether any one aerosol fraction carrying influenza virus retained infectivity longer at low RH. The amount of virus collected in the >4 µm aerosol fraction within the first 60 minutes of collection was approximately the same at 20% RH (1.3×104 virus) . Within 4 virus) . Continu4 virus) .6 virus) and 45% RH (1.2×106 virus)  . Within 6 virus) .5 virus) then at 45% RH (2.7×105 virus) (The amount of virus collected in the <1 µm aerosol fraction within the first 60 minutes of collection was more variable at 20% RH (5.8×105 virus) . However5 virus) . Continu5 virus) .Statistical analysis of the first 60 minutes showed there are significant main effects for humidity, fraction and time on virus infectivity and a significant humidity by fraction interaction. Specifically, with respect to humidity in general, infectious virus are reduced in the higher 45% humidity relative to low 20% humidity (p<0.0001). With respect to fraction, the number of infectious virus is highest in the 1–4 µM fraction and is significantly reduced in the <1 µM fraction and further reduced in the >4 µM fraction (p<0.0001). There was also a significant main effect of time (p<0.0068) with the first and last 15 minute collection intervals significantly lower than the two middle time points. The humidity by fraction interaction simply reflects that the size of the difference between the two humidity conditions varies as a function of fraction. Specifically, the smallest difference  was in the 1–4 µM fraction while the largest difference in the number of infectious virus was in the <1 µM fraction. However, there was no statistical difference in the rate of decay of infectious virus at 20% RH versus that at 45% RH in any of the 3 aerosol fractions once the initial loss in infectivity occurred within 0–15 min after coughing.The potential to transmit influenza by respirable aerosol particles (≤4 µm) is of particular concern as these particles can remain airborne for long periods and can be inhaled deeply into the lung to cause more severe infection Yang and Marr 3 viral particles <5 µm in size and a healthcare worker could potentially inhale up to 237 viruses. A dose-response model developed by Teunis et al. inf = 0.2–0.4) at low doses (101–2 TCID50 infectious units).Although most of the >4 µm particles were removed from the exam room at 4–5 h, a further decline in infectivity at 45% RH as compared to that at 20% RH nearly eliminates the potential for infection associated with particles of this size. Similarly, the potential for infection from influenza carried on the smaller particles was also further reduced at 45% RH, but the longer retention time of these particles in the air emphasizes the concern these sized particles still pose. The actual number of aerosolized viral particles that a healthcare worker could potentially inhale during a patient examination is largely dependent on the shedding rate of virus by the patient. Infected patients can shed 33 virus/min in aerosol particles ≥5 µm and 187 virus/min in particles <5 µm The effect of increasing humidity on viral survival differed among several reported studies as Hemmes et al. Extrapolation of Harper’s Hanley and Borup