Hantaan virus was discovered in Korea during the 1970s while other similar viruses were later reported in Asia and Europe. There was no information about hantavirus human infection in the Americas until 1993 when an outbreak was described in the United States. This event promoted new studies to find hantaviruses in the Americas. At first, many studies were conducted in Brazil, Argentina, Chile, Uruguay and Paraguay, while other Latin American countries began to report the presence of these agents towards the end of the 20th century. More than 30 hantaviruses have been reported in the Western Hemisphere with more frequent cases registered in the southern cone . However there was an important outbreak in 2000 in Panama and some rare events have been described in Peru, Venezuela and French Guiana. Since hantaviruses have only recently emerged as a potential threat in the tropical zones of the Americas, this review compiles recent hantavirus reports in Central America, the Caribbean islands and the northern region of South America. These studies have generated the discovery of new hantaviruses and could help to anticipate the presentation of possible future outbreaks in the region. Bunyaviridae. These viruses infect humans through inhalation of excreta from persistently and asymptomatically infected rodents [Hantavirus is a genus in the family  rodents . Some me rodents ,3.The viruses of this genus are oval or spherical particles with a diameter of approximately 100 nm . The hanApodemus agrarius. HTNV is considered the virus prototype of the new genus Hantavirus [The discovery of these viruses originated from a retrospective study of a HFRS outbreak during the Korean War 1950–1953). The etiologic agent was named Hantaan virus (HTNV), which was isolated from the wild rodent ntavirus ,9. Subse50–1953. Microtus pennsylvanicus in Maryland (United States) but no disease in humans has been associated with this agent [Peromyscus maniculatus was identified as the reservoir of SNV and the disease was named HPS [The first hantavirus identified in the new world was Prospect Hill virus (PHV) isolated from is agent ,10. Subsamed HPS ,11.The discovery of SNV promoted the search for the presence of these agents in other parts of the continent, and surveillance for possible new cases. This resulted in the discovery of many new hantaviruses that, at present, total more than 30 genotypes reported in the Americas. Most of these genotypes had been recovered from rodent hosts and have not been associated with human disease. However, sporadic cases of HPS have been diagnosed in Argentina, Peru, Brazil, Chile, Uruguay, Panama, Bolivia, Venezuela, Paraguay and French Guiana ,12,13 T. It has been proposed that hantavirus species must differ by more than 7% in their amino acid sequence of S and M segments. Although genotypes described so far differ widely in their degree of sequence similarity, in order to consider an isolate a new species by ICTV, a rigorous genetic, ecologic and antigenic characterization is required. Thus, some of the hantaviruses mentioned here are not real species but strains; in some reports a new strain of a hantavirus species was assigned a different name, such as Maripa virus, which is possibly a strain of Rio Mamore virus .Sigmodontinae rodents when the Isthmus of Panama was formed.A phylogenetic analysis of the S segment sequences shows that hantaviruses from South America and Panama are monophyletic with respect to those from North America . This suIt is worth mentioning that countries of the southern cone of South America  are at the forefront of hantavirus research in Latin America . AccordiOceomys bicolor rodent, captured in the center of Venezuela. Although not associated with human illness, it was observed that inoculation of a Syrian golden hamster (Mesocricetus auratus), with MAPV reproduced the symptoms of HPS, making this virus a model for the study of HPS pathogenesis [Since 1997, hantavirus circulation in Venezuela was evident with the isolation of Maporal virus (MAPV) from the ogenesis . Sigmodon alstoni rodent resulted in the discovery of another hantavirus named Caño Delgadito virus (CADV) [In 1997, a rodent sampling was performed on the Venezuelan grasslands in order to detect any hantaviruses. From this study, the analysis of viral sequences obtained from the cotton rat s (CADV) .In 1999, the first human case of hantavirus infection in Venezuela emerged, confirmed by serological tests for IgG and IgM, though information about the etiology of this particular virus is not available. Nevertheless, a human seroprevalence study was conducted after this report. This study analyzed 1,380 samples, from which 173 were collected in Caño Delgadito (Portuguesa), 367 in Caracas, 29 in Carabobo, 76 in Lara, 96 in Tachira and 64 from military recruits. Another 328 sera were collected from pregnant women from Anzoategui, and 246 more from Amerindian living in Zulia. The results displayed a prevalence of 1.7%, and the individuals from Carabobo showed a significantly higher seroprevalence than the entire population (10.3%) .Sigmodon alstoni behaved as a reservoir for CADV. In this case, rodents were inoculated with the viral isolate detecting viral RNA at 54 days post infection. It was also possible to recover infectious virions from different secretions, suggesting the development of a persistent infection and concluding that S. alstoni acts as a reservoir for this virus [There has also been some research on the characterization of the interactions of these two viruses with their hosts. One particular study evaluated whether is virus .Sigmodon alstoni rodent and all sequences from Oligoryzomys sp grouped with the MAPV. There were no sequences of these two viruses present in any other species, suggesting that these two rodents are the main hosts for both viruses [Oligoryzomys genus and accordingly, in a subsequent study, they analyzed the Cytochrome b sequences of Choclo virus (CHOV)-infected Oligoryzomys rodents from Panama, and MAPV-infected Oligoryzomys from Venezuela. This work found that Oligoryzomys costaricensis is associated with the CHOV and Oligoryzomys delicatus is associated with the MAPV [Years later, a study obtained viral sequences from 280 rodents captured on farms in western Venezuela, all the sequences corresponding to the CADV originating from the  viruses . In thisthe MAPV .Sigmodontinae subfamily [In 2004, the first report supporting evidence of hantavirus circulation in Colombia was published. This work evaluated the presence of reactive IgG against the SNV antigen in a population of rural workers from the states of Cordoba and Sucre, and detected a seropositivity prevalence of 13.5% . The samubfamily . et al., carried out a study in northwestern Antioquia department where they captured 354 rodents and evaluated for the presence of IgG reactive for SNV and Maciel virus (MCLV), finding 15 positive rodents belonging to the species Zygodontomysbrevicauda cherriei [Later, Londoño cherriei . Tissuescherriei .Myodes glareolus. Since this rodent has not been detected in the Americas, the authors concluded that this patient was possibly infected on his return to Belgium [Despite previous reports in neighboring countries, no studies in Ecuador indicate circulation of these viruses. The only publication speculating its presence is the case of a Belgium tourist who developed epidemic nephropathy from hantavirus after his return from a trip to Ecuador. In that patient, it was initially thought that he was infected by the Seoul virus (SEOV), the only old world hantavirus whose circulation has been established in the new world . Later o Belgium .Oligoryzomys microtis [In Bolivia, hantavirus circulation was evident in 1996 when the Rio Mamore virus (RIOMV) was described through characterization of sequences obtained from the rodent microtis . In 1998microtis ,48.Oligoryzomys chaconesis in Oran city . Although the second patient lived in Bolivia, he was working as a muleteer in Salta province (Argentina) and returned to Bolivia three weeks before symptoms appeared; therefore it is unclear where he acquired the infection [Between May and July of 2000, two human cases of HPS in Bermejo City were confirmed by IgG and IgM ELISA assay and RT-PCR. Viral sequences obtained from the first patient indicated that the etiological agent belonged to a previously reported Andes North linage , and thonfection . et al., in 2005 described the presence of hantavirus antibodies in the rodent Oligoryzomys microtis captured in Mineiros and Calomys callosus captured in Concepcion [O. microtis showed 90% identity with RIOMV and those from C. callosus showed 87% to 88% identity with LANV. Those obtained from the sample of the second patient showed 99% identity with those obtained from C. callosus, indicating that this rodent is the host for LANV, responsible for the HPS case near Concepcion. The samples of patients from Mineiros were not available for comparison with the rodent sequence. This result is consistent with the hypothesis that C. callosus y C. laucha (the reservoir of LANV in Paraguay) show a high genetic similarity, potentially indicating that this virus evolved from a common ancestor [In August 2002, two human cases of HPS were confirmed in Mineiros and Concepcion in the state of Santa Cruz. A study carried out by Carroll, ncepcion . The autancestor . et al., sampled febrile patients suspected of having suffered recent hantavirus infection in Chapare Province in 2008–2009 [With the goal of performing a better description of the genetic diversity of Bolivian hantaviruses, Cruz 008–2009 . Viral R008–2009 . In summOligoryzomys microtis rodents captured in Iquitos, capital of the Maynas Province . This virus was originally called HTN-007 [In 1996, a variant of RIOMV was found in  HTN-007 , though  HTN-007 . Sequenc HTN-007 .In 2011, two fatal cases with clinical suspicion of HPS and HFRS occurred in Loreto, in the Amazon region of Peru. The first case was a 29-year-old woman, who visited locations along the Nanay River working as a tourist guide. This person showed fever, headache, and acute pulmonary edema requiring hemodialysis days before her death. The second case was a 33-year-old woman who died in a hospital in Iquitos; she developed fever, headache, myalgia, hemorrhagic manifestations, acute renal failure with liver involvement, and cardiovascular and lung damage. In both cases the diagnoses were confirmed by the detection of IgM and RT-PCR amplification of 540nt of the M segment, which showed 97% homology with the SEOV . This agOligoryzomys microtis in the same location [Two additional cases of HPS were reported that year near the city of Iquitos; both were clinically characterized by fever, malaise, abdominal pain, watery diarrhea, coughing, cyanosis and severe acute respiratory failure, which evolved to renal failure and refractory shock. In both patients the causative agent was confirmed by molecular tests, which demonstrated 96% similarity with the RIOM variant previously detected in location .A retrospective study was performed on febrile patients tended to in 12 health centers from 2007 to 2010 in the Maynas province in order to find serological evidence of recent hantavirus infection. Fifteen out of 5,174 patients, who had suffered a relatively mild disease, were found to be IgM reactive to ANDV antigen. In the same study, a cross-sectional analysis of healthy residents of Iquitos revealed a seroprevalence of 1.7% (33/2063) .The presence of hantavirus circulation in French Guiana was suggested in a retrospective serologic survey which evaluated the seroprevalence in 420 patients with compatible symptoms with HPS, finding that seroprevalence of IgG was 1.42% and none of the samples were positive for IgM . A HPS cAccording to the scientific literature, increasing numbers of studies performed in recent years in Brazil report on the circulation of hantavirus. In this review we will focus only on those studies conducted in the northern and Amazonian region of Brazil.The first case of HPS in Brazil was reported in 1993 in the state of São Paulo; the causative agent was identified as Juquitiba virus (JUQV) . Other cet al., conducted a genetic analysis of viral sequences from patients and rodents captured in the presumed sites of infection [Bolomys lasiurus and Oryzomys nigripes were the hosts of ARAV and JUQV, respectively [By 2004, the number of diagnosed cases of HPS in Brazil had increased to 342, most being located in the south and southeast of the country. Some cases were also found in the sates of Minas Gerais (54), Mato Grosso (33), Maranhao (7), Para (4), Goias (3), Rio Grande do Norte (1) and in Bahia (1) . Considenfection . Partialectively .In the Amazon region of Brazil, circulation of hantavirus was reported in 2004; the first cases of HPS were reported in four patients, all inhabitants of a rural area near Itacoatiara. All of these patients belonged to same family that had cleared a forest near their farm and were declared to have been in contact with many rodents. All of the cases but one were confirmed by the IgM ELISA test .Oligoryzomys fornesi and Holochilus sciureus, respectively [A cross-sectional study in Maranhão between 2004 and 2006 evaluated potential risk factors for hantavirus infection in this region. Samples were obtained from 1,389 inhabitants from this area and tested for IgG presence against SNV antigen. The seroprevalence detected was 4.7%, with most of the positive samples from farmers . In the ectively .Oligoryzomys utiaritensis [Another study published in 2010 estimated the seroprevalence of antibodies reactive to a recombinant capsid antigen of ANDV in different areas in the Amazon area and found 2.16% in Novo Progresso, 4.37% in Trairão (state of Para), 4.74% in Guarantã do Norte and 9.43% in Marcelândia (state of Mato Grosso). The Other studies confirmed the circulation of CASV virus in samples of patients with HPS through viral genome amplification and detect its likely principal rodent host ritensis ,58.The human seroprevalence for hantavirus in the Amazon state was estimated with samples collected from 2007 to 2009. This was determined by ELISA assay using recombinant nucleocapsid protein from ARAV. About 0.6% (10/1731) of the samples were positive for IgG and none of the same samples were positive for IgM. The positive individuals belonged to the municipalities of Atalaia do Norte, Careiro Castanho, Itacoatiara and Labrea indicating exposure to hantavirus throughout the entire state .Oryzomys Couesi and Sigmodon mascotensis identified in Colima, southwestern Mexico, and the El Moro Canyon virus (ELMCV) carried by Reithrodontomys megalotis, initially detected in the United States [Reithrodontomys mexicanus, Reithrodontomys sumichrasti, Peromyscus maniculatus, and Peromyscus levipes [Unlike the New Mexico panorama where the first HPS cases reported resulted in the discovery of SNV, Mexico has not registered any case of HPS despite various studies conducted in its territory . This sid States ,60. Addi levipes ,62.Peromyscus beatae, Megadontomys sumichrasti, and Reithrodontomys megalotis, respectively [Neotominae subfamily and 10 species from Sigmodontinae), sampled from eight different states. The seroprevalence in this study was 4.0% and samples from seropositive rodents were used to amplify viral genomes. Five rodents were positive for SNV sequences indicating that this virus is enzootic in Nuevo León, San Luis Potosí, Tamaulipas and Veracruz states. Viral sequences obtained from Reithrodontomys megalotis and R. sumichrasti indicated ELMCV circulation, and sequences obtained from Peromyscus spicilegus, P. melanotis, P. hylocetes, P. levipes and P. ochraventer indicated the presence of Limestone Canyon (LSCV) virus and other hantaviruses were widely distributed throughout Mexico [Recently genetic evidence of hantavirus circulation in Mexican rodents was published. The first study captured 211 rodents in Guerrero and Morelos states and reported three new hantaviruses: Montano (MTNV), Carrizal (CARV) and Huitzilac viruses (HUIV) in ectively . The sect Mexico .Oligoryzomys fulvescens rodents captured in the same place. The viral sequence obtained from these rodents had a high similarity to the one obtained from patients with HPS, indicating that this rodent is a host for CHOV. Additional sequences were obtained from Zygodontomys brevicauda, but these belonged to another hantavirus, which was later named the Calabazo virus [In 1999, suspected HPS cases were reported in the Azuero peninsula, from patients whose diagnostics were confirmed by serologic testing and viral genome detection. Sequences obtained from S and M segments were used in phylogenetic analysis and indicated that these patients were infected with a new hantavirus denominated as Choclo virus (CHOV). This was the first evidence of pathogenic hantavirus circulation in this country . Subsequzo virus . One impzo virus .Oligoryzomys fulvescens and Zygodontomys brevicauda in the Azuero peninsula were 0.06% (4/72) and 0.05% (5/108) respectively suggesting that the former could be the principal host for CHOV and the Calabazo virus [Oligoryzomys costaricensis and not Oligoryzomys fulvescens as previously mentioned [A further study was performed to identify populations of small mammals with potential importance in the epidemiology of HPS in Panama. Researchers sampled 556 rodents and marsupials from central and western Panama from 2000 to 2002 and evaluated the presence of antibodies (IgG) against hantavirus. The results indicated that 2.7% of animals were positive. This study also reported sequences of another hantavirus previously unidentified in Panama, that was called Rio Segundo virus (RIOSV). Seroprevalences for hantavirus in zo virus . Conversentioned .et al., performed several hantavirus seroprevalence studies in different communities: three in the Santos province and one in the Veraguas providence, and the prevalence of these agents varied between 16.5% and 60.4% [An ecological study also demonstrated that the occurrence of the Panamanian outbreaks was favored by agricultural activity, especially for rice production, and the biodiversity reduction of wild rodents, supporting the increase of the population density in the rodents known as hantavirus main hosts ,65. Fromnd 60.4% .Except for Panama, most of Central American countries have not performed sufficient studies regarding hantavirus circulation. Some countries have limited information and others have not yet begun this work. Reithrodontomys mexicanus from Costa Rica [Oryzomys couesi seropositive for hantavirus. In this study, viral isolation from lung tissue of one seropositive rodent was successful and the agent was characterized as a new virus closely related to Bayou virus (BAYV) and named the Catacamas virus (CATV) [Research in Central America began in 1995, and soon after the discovery of SNV, a new Hantavirus called RIOSV was identified in rodent samples of sta Rica . Anothers (CATV) .Rattus norvegicus, an identified SEOV host, indicating that SEOV or other related hantaviruses might be present in Barbados [Evidences of hantavirus circulation has been mostly limited to the continent. The first report from a Caribbean island was published in Barbados in 2002. This research found hantavirus IgM and IgG serological evidence in patients with suspected leptospirosis. Later serological evidence was also detected in Barbados . In TrinBarbados .During recent years, the understanding and diagnoses of hantavirus infections have improved notably in the Americas. Unlike the southern cone of South America and Brazil that have developed many studies about hantavirus circulation immediately after the discovery of SNV , other tSupporting this affirmation, many studies have reported serological evidence of Hantavirus circulation in rodents and humans, the first one showed a broad diversity in likely primary hosts in these countries. This emphasizes the importance of improving the knowledge about the ecology of these species and characterizing human activities that generate risk to get hantavirus infection . In addiAlthough there are many studies that provide some evidence for new hantaviruses, most reports have not carried out the assays that fulfill the requirements of the International Committee for the Taxonomy of Viruses (ICTV) to define a new species. These conditions are: (I) a divergence of the amino acid sequence higher than 7% in comparison with the previously reported viruses that is supported by the complete sequences of their segments S and M; (II) at least four-fold difference on a cross sero-neutralization assay; (III) the new virus candidate must not be a product of genetic reassortment between different previously identified hantavirus strains; and (IV) the new virus should occupy a unique ecological niche with a specific primary reservoir . StudiesThis landscape would allow us to determine whether the frequency of HPS infection is truly increasing in Latin America. It is noteworthy that the lack of clinical cases in some countries is directly proportional to the absence of research performed on this area. This is probably the case in most of the tropical countries of Central and South America, since HPS and HFRS are not considered a differential diagnosis in compatible human cases, and also because of the lack of diagnostic tools needed to confirm the suspected cases. As a consequence, new studies are needed that improve the knowledge about the dynamic of circulation of these new viruses that allow us to reveal possible new hantaviruses and to increase the awareness among medical care centers, physicians and other health care professionals. We also need studies that help us to build infrastructure for research on this topic in developing countries, which also happen to be the places where most emerging viruses are thriving , and ide


To the Editor: West Nile Virus (WNV) is rapidly spreading in the Western Hemisphere , which includes Saint Louis encephalitis virus (SLEV) (in North and South America), Japanese encephalitis virus (Asia), and Murray Valley encephalitis virus  . None ofThese serologic data should be considered indirect evidence of WNV activity in Colombia. We encourage Colombian human and animal health authorities to enhance surveillance for human, equine, and avian disease attributable to WNV. Efforts are needed to isolate the virus or detect specific viral RNA to confirm this finding and to identify vectors and vertebrate hosts involved in WNV transmission in Colombia.


Large-scale vaccination of animals might have influenced virus evolution. Phylogenetic relationships were examined for 198 Rift Valley fever virus isolates and 5 derived strains obtained from various sources in Saudi Arabia and 16 countries in Africa during a 67-year period (1944–2010). A maximum-likelihood tree prepared with sequence data for a 490-nt section of the Gn glycoprotein gene showed that 95 unique sequences sorted into 15 lineages. A 2010 isolate from a patient in South Africa potentially exposed to co-infection with live animal vaccine and wild virus was a reassortant. The potential influence of large-scale use of live animal vaccine on evolution of Rift Valley fever virus is discussed. Bunyaviridae and genus Phlebovirus. Large outbreaks occur at irregular intervals when heavy rains favor breeding of mosquito vectors of the virus and are characterized by deaths of newborn animals and abortion in pregnant sheep, goats, and cattle. Humans become infected by contact with tissues of infected animals or from mosquito bites and usually show development of mildly to moderately severe febrile illness. However, severe complications, including ocular sequelae, encephalitis, and fatal hemorrhagic disease, occur in some patients  is an acute disease of domestic ruminants in Africa and the Arabian Peninsula. This disease is caused by a mosquito-borne virus of the family Rift Valley fever virus (RVFV) has a negative-sense, single-stranded RNA genome comprising large (L), medium (M), and small (S) segments. The L segment encodes viral RNA polymerase. The M segment encodes envelope glycoproteins Gc and Gn, a nonstructural protein, and a 78-kDa fusion protein of nonstructural and Gn proteins. The S segment shows an ambisense strategy and encodes nucleocapsid protein N and a nonstructural protein  according to the manufacturer’s instructions.A 10-μL RNA aliquot was analyzed by using reverse transcription PCR with the Titan One Tube RT-PCR Kit  in a final volume of 50 μL as described  according to the manufacturer’s instructions. Nucleotide sequences of PCR products were determined by using BigDye version 3.1 Terminator Cycle Sequencing Ready Reaction Kits  according to the manufacturer’s instructions. For confirmatory purposes, sequences were obtained for both strands of PCR products by using primers FD1 and RVF E. Products were purified by using CentriSep spin columns  and analyzed by using a 377 GenAmp Sequencer and a 3130xl Genetic Analyzer .On the basis of findings for M segment data, partial sequences were determined for S and L RNA segments of isolate SA184/10 from a patient potentially co-infected with live animal vaccine and wild virus; isolate SA54/10 from another patient in the same area; the batch of vaccine used (Smithburn neurotropic strain [SNS] 105/2010); SNS vaccine master seed virus; 95EG vaccine; and historical isolates 95EG Cow-2509, H1739, and H1825  to test Nucleotide and amino acid sequence alignments were generated by using ClustalW Multiple Alignment analysis software as implemented in BioEdit version 7.0.5.3  contained single isolates, and lineage I contained 2 isolates from South Africa. Six other lineages  contained clusters of isolates associated with outbreaks in individual countries or regions, but each also included isolates from distant locations or separate outbreaks. Thus, lineage A comprised isolates from outbreaks in Zimbabwe in 1978, Madagascar in 1979, and Egypt in 1977–1978 and 1993 and a 1974 isolate from an interepidemic period in Zimbabwe .,Lineage C was most widely distributed and contained isolates from major outbreaks in Zimbabwe in 1978, Madagascar in 1991, eastern Africa  during 1997–1998, Saudi Arabia during 2000–2001, Kenya in 2007, and South Africa during 2008–2009 and lesser outbreaks and interepidemic periods in the same countries and Mauritania during 1976–2009. It was also associated with an isolated fatal infection in Angola in 1985 in a visitor from South Africa ostensibly exposed to mosquito bites  of 490 nt (2 aa), and distantly from 46 other human isolates from the 2008–2010 outbreaks in South Africa. Moreover, isolate 95EG Cow-2509 from the fetus of a cow that aborted after the administration of 95EG vaccine in Egypt grouped in lineage L distantly from all other isolates from Egypt in lineage A . IsolatePartial nucleotide sequence data for S, M, and L RNA segments derived from whole-genome sequences of 33 isolates produced phylogenetic trees with the same topologies as those published for the complete sequences (,Historical developments in RVF include spread beyond sub-Saharan Africa during 1977–2007 and fatal human infections during a large outbreak in South Africa during 1974–1976. Since that time, large outbreaks of the disease in livestock have invariably been associated with human deaths (Recent outbreaks in South Africa showed an analogous pattern. After heavy rains in 2008, lineage C virus, which had been isolated during a limited disease outbreak in Kruger National Park in 1999, was associated with scattered outbreaks of RVF in adjacent parts of northeastern South Africa. In the first half of 2009, the same lineage caused limited outbreaks to the south in KwaZulu-Natal Province. In the second half of 2009, lineage H virus, which had been encountered in the Caprivi Strip of Namibia in 2004, caused focal outbreaks in Northern Cape Province and was progressively identified in coalescing outbreaks over much of interior South Africa in 2010 . Diversi,––,,,Reassortment between SNS vaccine virus and wild virus has implications for the safety of the vaccine and its possible role in evolution of RVFV. The SNS virus from Uganda  was take,During 1951–1968, ≈1 million doses of SNS animal vaccine were sold in South Africa; sales were similar in Kenya ,. Sales d,,,,During the same period, 4.7 million doses of newly developed inactivated RVF vaccine and ≈14 million doses of Wesselsbron virus vaccine were sold (Livestock vaccines are sold in multidose vials and commonly administered with automatic syringes and intermittent changes of needles. Vaccine is likely to be administered to some animals that are already infected. Thus, in view of intense viremia that occurs in RVF, serial transfer of wild virus by needle is a recognized hazard of vaccinating livestock during outbreaks (Although there is no corresponding evidence that reassortment or recombination occurred on a large scale, few isolates remain available from the 1969–1970 and 1974–1976 outbreaks in Zimbabwe and South Africa. Nevertheless, multiple new lineages were encountered during 1969–1981, particularly in Zimbabwe where intensive monitoring was instituted during 1971–1979 and where lineage A and C isolates were obtained . Moreove,Thus, the partial M segment nucleotide sequence of reassortant isolate SA184/10 differs from that of vaccine virus, and that of isolate 95EG Cow-2509 from the aborted fetus of a vaccinated cow differs even more markedly from that of vaccine virus and from all other known isolates from Egypt but has some relationship with isolates H1739 and H1825 in lineage L , which cNew vaccines, some with natural or induced attenuating deletions or mutations in all 3 segments of the genome, are being developed (


To the Editor: Certain arthropod-borne epornitic flaviviruses, namely, West Nile virus (WNV) and Usutu virus (USUV), have spread recently in parts of Europe (Alectoris rufa) and 13 common pheasants (Phasianus colchicus) were collected on 12 hunting properties from Cádiz  , BAGV (p<0.0001), and USUV (p<0.0001) . The sigNeutralizing antibodies to >1 flavivirus were detected in 15 of the 45 VNT-positive partridges and in 6 of the 12 VNT-positive pheasants . SpecifiAnalysis of VNT results in juvenile partridges showed specific neutralizing antibodies to WNV (13%) or BAGV (9%); 4% of these samples were positive for flavivirus but inconclusive for any of the flaviviruses tested . OverallEvidence of infection by at least WNV and BAGV in 2 consecutive seasons strongly supports the premise that these viruses overwintered in the area. Capability of WNV to overwinter in southern Europe was shown in Italy during 2008–2009 (Study area in Spain (province of Cádiz) showing the position of hunting properties analyzed.


Herpes simplex virus type 1 is a neurotropic herpesvirus that establishes latency within sensory neurones. Following primary infection, the virus replicates productively within mucosal epithelial cells and enters sensory neurones via nerve termini. The virus is then transported to neuronal cell bodies where latency can be established. Periodically, the virus can reactivate to resume its normal lytic cycle gene expression programme and result in the generation of new virus progeny that are transported axonally back to the periphery. The ability to establish lifelong latency within the host and to periodically reactivate to facilitate dissemination is central to the survival strategy of this virus. Although incompletely understood, this review will focus on the mechanisms involved in the regulation of latency that centre on the functions of the virus-encoded latency-associated transcripts (LATs), epigenetic regulation of the latent virus genome and the molecular events that precipitate reactivation.This review considers current knowledge and hypotheses relating to the mechanisms involved in the establishment, maintenance and reactivation herpes simplex virus latency. Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are closely related ancient human pathogens responsible for a number of diseases of minor, moderate and severe pathology including oral and genital ulceration, virally induced blindness, viral encephalitis and disseminated infection of neonates. HSV-1 is typically transmitted during childhood, whilst HSV-2 is the cause of most genital herpes . WorldwiHSV is a neurotropic virus and it is within sensory neurons that latent infection is established. Upon primary HSV replication at the oral or genital mucosa, the virus is able to infect the neuronal dendrites of sensory ganglia that innervate these tissues, and after retrograde microtubule-associated transport to the nerve cell body, the virus encounters a ‘choice' of gene expression programmes that dictates the fate of the neuron: lytic replication or latent infection. The latent virus genome is stably retained within sensory neurones and is characterized by repression of all viral lytic genes. In response to a variety of diverse stimuli, the virus can periodically reactivate to resume virus replication and produce infectious virus, which is transported anterograde back to the periphery to facilitate epithelial cell infection and consequent transmission.in vivo, for example, by transient induction of hyperthermia  expressed and can the virus be easily reactivated.1 or γ-1), DNA replication and finally late (L2 or γ-2) gene expression  genes and then proceeds through early (E or β), ‘leaky-late’  class 1 evasion gene), all of the IE proteins are involved in gene regulation. Two of these proteins, ICP0 and ICP4, are both major transactivators of gene expression. ICP0 does not bind DNA and is a promiscuous transactivator of gene expression . Althoug+ T cell recognition via ICP47-mediated TAP inhibition  or RT-PCR methods  of viral DNA . In particular, virus-encoded miRNAs are found in the majority of herpesviruses studied to date . MiR-H1 et al. utilized cell lines that encoded, a region of LAT in which no miRNA proposed to target ICP0 or ICP4 is found. However, within this region, two sRNA species have recently been discovered  epitope and not IE proteins. gB is a leaky-late gene and, the fact that the reactivating neuron was recognized by a gB-specific CD8+ T cell indicates that IE and early genes are likely to be expressed prior to a CD8+ T cell-mediated block in reactivation. These data indicate that, not only are neurons able to survive high viral genome inputs, they can also survive extensive viral gene expression  and each is flanked by inverted repeats , these preferential sites swapped between viruses, defining the LAT as the viral determinant of this specificity . Given that approximately 50% of all neurones latently infected with HSV-1 are A5+  reactivate and are antigen-positive 22-h poststimulus  responsible for the initiation of the lytic gene expression and exit from latency. ICP0 has always been regarded as the most likely candidate owing to its ability to target ND10 domains for degradation and its capacity to act as a promiscuous activator of gene expression  treatments from latently infected PC12 cells revealed that only mutants lacking functional VP16 failed to reactivate (A recent publication by Thompson and Sawtell (nfection  in up tonfection . Recent nfection . Experim+ T cell immunosurveillance may influence both the efficiency and mechanism by which reactivation can occur (Reactivation from latency is a critical phase of HSV life cycle; therefore, it is not surprising that it is highly regulated. It seems that only a small proportion of latently infected neurones are able to respond to a given reactivation stimulus at any given time. The responsiveness of latent virus genomes may be related to the nonuniform nature of the latent state. Thus, multiple factors such as latent DNA copy number, level of LAT expression, neuronal type, degree of genome repression and CD8an occur . Given t


Variola virus, the agent of smallpox, has a severely restricted host range (humans) but a devastatingly high mortality rate. Although smallpox has been eradicated by a World Health Organization vaccination program, knowledge of the evolutionary processes by which human super-pathogens such as variola virus arise is important. By analyzing the evolution of variola and other closely related poxviruses at the level of single nucleotide polymorphisms we detected a hotspot of genome variation within the smallpox ortholog of the vaccinia virus O1L gene, which is known to be necessary for efficient replication of vaccinia virus in human cells. These mutations in the variola virus ortholog and the subsequent loss of the functional gene from camelpox virus and taterapox virus, the two closest relatives of variola virus, strongly suggest that changes within this region of the genome may have played a key role in the switch to humans as a host for the ancestral virus and the subsequent host-range restriction that must have occurred to create the phenotype exhibited by smallpox. Poxviridae consists of a very diverse set of viruses that share the ability to replicate in the cytoplasm of host cells and a linear dsDNA genome structure. This diversity manifests in a variety of ways: 1) genome size ranges from 134–360 kbp; 2) G+C genome composition ranges from 35–78%; 3) host range for the family as a whole is extremely wide ; 4) host range for a particular virus maybe broad (e.g. cowpox virus (CPXV)) or narrow ); 5) infection may be acute (e.g. most poxviruses) or persistent (e.g. molluscum contagiosum virus (MCV)).The taxonomic family Without a traditional fossil record, it is difficult to understand the evolutionary pathways of viruses. However, the availability of new host organisms, through evolution or population growth, can play a pivotal role in the evolution of new virus species. Measles virus is believed to have evolved from a rinderpest virus 1000–1500 years ago One of the most interesting questions in the evolution of the orthopoxviruses is the origin of smallpox, which, over the centuries, may have killed as many as 300 million people; the restricted host range  and disease severity  are two of the most salient features of this virus Various phylogenetic analyses indicate that VARV, camelpox virus (CMLV) and taterapox virus (TATV) are sole members of an orthopoxvirus clade; these methods Recombination is the scourge of phylogenetic studies, and since it is known that recombination has played a significant role in the evolution of at least some poxviruses The genomes used for the construction of the MSA were: HSPV-MNR76, DQ792504; VACV-CVA, AM501482; VACV-WR, NC_006998; VACV-Lister, AY678276; VACV-Cop, M35027; RPXV-Utr, AY484669; CPXV-AUS_1999, HQ407377; CPXV-GRI, X94355; CPXV-FIN_2000_MAN, HQ420893; CPXV-GER_1980_EP4, HQ420895; CPXV-GER_2002_MKY, HQ420898; CPXV-GER91, DQ437593; CPXV-GER_1998_2, HQ420897; CPXV-GER_1990_2, HQ420896; CPXV-FRA_2001_Nancy, HQ420894; CPXV-NOR_1994_MAN, HQ420899; CPXV-BR, NC_003663; CPXV-UK2000_K2984, HQ420900; TATV-DAH68, NC_008291; CMLV-CMS, AY009089; VARV-GBR44_harv, DQ441444; MPXV-ZAR, NC_003310; ECTV-Mos, NC_004105. Sequences were aligned using MAFFT The Find Differences tool in BBB was used to compare groups of user-selected sequences in an MSA; it identifies those columns (nucleotide positions) in the MSA that satisfy the following conditions: nucleotides must be identical in all of the sequences in group 1 (All the same column), different from all sequences in group 2 (All different column), optionally, additional groups of either column (All the same or All different) can be added to the comparison.The tolerance parameter was added to the BBB software to allow differences to be found when only 1, 2, or 3 genomes in a large alignment fail to satisfy the Find Differences search. For example, if VARV, CMLV and TATV have a unique block of sequence that is not present in 18/20 of the other genomes in the MSA then Find Differences will ignore the other 2/20 that have the unique block of sequence and print out the names of the “tolerated genomes” with the found positions. The tolerance parameter can be set for the all same or the all different group of viruses in these searches.A neighbor-joining (NJ) tree was constructed to perform phylogenetic analyses, as did Qin et al. We tested the statistical significance of SNP clustering by binning the data and then calculating a p-value for the number of SNPs in each bin under a null hypothesis of a Poisson distribution. In order to ensure this result was robust to binning parameters we tested different bin sizes  as well as start positions , which had no effect on the significance of the peak we observe (data not shown). We also used a non-parametric method in which we randomly permute the SNPs throughout the region 1,000,000 times and record the bin with the largest number of SNPs and used this distribution to determine a significance cutoff; this gives us the same result as the parametric method (data not shown).smallpox-defining changes. Therefore, since CMLV and TATV, which form a unique clade with VARV, also have very restricted host ranges, we decided to take a different approach and focus on the SNPs common to these three viruses and different in all other orthopoxvirus genomes within our test group. An important feature of this analysis is that it is not confounded by the SNPs arising post-speciation of VARV, CMLV and TATV. A multiple alignment of the genomes was constructed using MAFFT Individual VARV genomes have approximately 700 unique SNPs when compared to the common ancestor with CMLV and TATV, and when compared to individual CPXVs, the pairs of genomes have approximately 2–3,000 SNPs  representing changes in both CPXV and VARV genomes from the most recent common ancestor ; this number is too large to identify potential all the other orthopoxvirus genomes in the alignment? This was initially performed looking at 1 genome of each species and subsequently confirmed with the other members. Such changes should have occurred during evolution from last common ancestor of the VARV+CMLV+TATV clade and the other orthopoxviruses to the separation of VARV and CMLV+TATV (First we asked which nucleotide positions (SNPs) are identical in VARV+CMLV+TATV and different in MLV+TATV ; a similMLV+TATV . This anMLV+TATV , methodsMLV+TATV . In ordeexcept 1, 2 or 3 tolerated genomes?” The tolerances of 1 and 2 had a very pronounced effect on the spectrum of SNPs associated with the VARV+CMLV+TATV clade, increasing the number within the O1L ortholog by 14 and 28, respectively, with relatively little effect elsewhere in the genome. However, the effect was also remarkable for 1) the particular viruses that were matched to particular SNPs were almost exclusively CPXVs, and 2) the SNPs associated with the tolerated viruses clearly appeared as related groups. Examples include: 1) CPXV-GER1980 is the single tolerated virus in 6 of 7 SNPs within a 70 bp region, 2) CPXV-GER1980 and CPXV-AUS1999 are the two tolerated viruses over a 34 bp region with 8 SNPs and 3) for CPXV-NOR1994, 4 SNPs are tolerated over a 33 bp region, but 2 are also tolerated with CPXV-BR and the other 2 are tolerated with CPXV-FIN2000  the gene is completely deleted from Yaba monkey tumor virus YMTV; ; 2) threAll similarity and motif searches failed to detect any non-poxvirus matches. However, it was interesting to note that the O1L protein orthologs are very diverse with percent aa identity values resembling those for the intracellular enveloped virus (IEV) protein F12L  it is relatively poorly conserved compared to core proteins such as the RNA polymerase subunit RPO147 The SNP data analysis presented here indicates that an unusually high number of changes were introduced into the O1L ortholog after creation of the VARV+CMLV+TATV lineage but before VARV speciation. The origin of this gene is ancient, and although it is present in most members of all The data presented here supports previous hypotheses regarding the important role that recombination plays in the evolution of the poxviruses Finally, because the VARV O1L ortholog has been retained in all smallpox viruses and the host range of smallpox is strictly limited to humans, it is reasonable to assume that that the gene has undergone subsequent selection for optimal function in human cells and may in fact be important for the replication/infection cycle of VARV; however, how this relates to the virus case fatality rate cannot be predicted. Since deletion of the O1L gene from VACV-CVA only made the virus sensitive to drugs targeting the epidermal growth factor receptor (EGFR) in CV-1 (monkey) cells and not CEF cells Text S1The file contains the multiple sequence alignment of genome cores, after gaps have been removed. The sequences are in FASTA format. The file can be loaded into the Base-By-Base software.(FASTA)Click here for additional data file.


We read with great interest the recently published article by Ilesanmi and colleagues in your journal . The autThe number of published studies that related patient costs of hypertension treatment in Nigeria is quite low despite the high burden of the disease . There iIn order to appropriately design future studies, we would like to present some further points that the study did not address. Although the authors highlighted in the background of their paper that direct costs constituted treatment-related costs and indirect costs constituted the lost man hours/savings among workers; it reported indirect costs as costs due to transportation which indeed should be part of the direct costs. Future studies should clearly define direct costs to include the out-of pocket (monetary) expenditures such as payments for administrative fees, drugs, laboratory tests, transport, hospitalization and food; while indirect costs defined as time loss of income such as transportation time, waiting times, absence from work. There is need to also document patients’ costs of coping with the hypertension issues like loans and, sale of assets .About two-third 167 (66.8%) of the study participants were aged 60 years and above. This clearly indicates that the proportion having elevated blood pressure is more of the late middle age and elderly group. It also suggests that at this age they are more likely to visit the hospital with a family supporter (member). Such family supporters also incur additional direct  as well as indirect  costs: these needs to be considered in other to determine the full household costs of care per visit .Household catastrophic expenditures for care have commonly been described as costs exceeding 10% of income . Using tDespite being a monotherapy, alpha-methyl dopa emerged as the most cost effective drug in the cohort of patients studied. It is not clear if the stage of the hypertension determined the type of anti-hypertensive medication given to the patient. It is possible that patients with mild hypertension were more likely to receive monotherapy of alpha-methyl dopa which easily controlled the blood pressure. It is also possible that for patients who received alpha-methyl dopa-based antihypertensive combination therapy, the additional antihypertensives were introduced after a failure of the monotherapy. Previous studies have shown that drug class affected blood pressure'lowering efficacy in patients with differing severities of hypertension .Post-diagnosis, Nigerian patients may require three to five re-treatments visits within 6 months to achieve target blood pressure levels . The stuFinally we would like to thank the authors for such an important study. We feel that their results should attract the attention of policy makers for better control of hypertension in Africa.


Narcissus pseudonarcissus) are one of the world's most popular ornamentals. They also provide a scientific model for studying the carotenoid pigments responsible for their yellow and orange flower colours. In reverse bicolour daffodils, the yellow flower trumpet fades to white with age. The flowers of this type of daffodil are particularly prone to colour break whereby, upon opening, the yellow colour of the perianth is observed to be 'broken' into patches of white. This colour break symptom is characteristic of potyviral infections in other ornamentals such as tulips whose colour break is due to alterations in the presence of anthocyanins. However, reverse bicolour flowers displaying colour break show no other virus-like symptoms such as leaf mottling or plant stunting, leading some to argue that the carotenoid-based colour breaking in reverse bicolour flowers may not be caused by virus infection.Daffodils (Gomphrena globosa, suggesting the presence of potexvirus. Red necrotic lesions were not observed in this indicator plant when sap from reverse bicolour flowers not showing colour break was used. The reverse transcriptase polymerase reactions using degenerate primers to carla-, potex- and poty-viruses linked viral RNA with colour break and sequencing of the amplified products indicated that the potexvirus Narcissisus mosaic virus was the predominant virus associated with the occurrence of the colour break.Although potyviruses have been reported to cause colour break in other flower species, enzyme-linked-immunoassays with an antibody specific to the potyviral family showed that potyviruses were not responsible for the occurrence of colour break in reverse bicolour daffodils. Colour break in this type of daffodil was clearly associated with the presence of large quantities of rod-shaped viral particles of lengths 502-580 nm in tepals. Sap from flowers displaying colour break caused red necrotic lesions on Narcissus mosaic virus was the virus that was clearly linked to the carotenoid-based colour break.High viral counts were associated with the reverse bicolour daffodil flowers that were displaying colour break but otherwise showed no other symptoms of infection.  Colour breaking of flowers is a long-documented type of variegation in the flower where the usual pigment pattern of the perianth is changed to irregular patches or streaks of pigmentation. Colour break occurs in many plant species including camellia , orchidsColour break in tulips is unpredictable and its cause was not known in the seventeenth century, although grafting experiments in 1637 demonstrated that the trait could be transmitted from bulbs of variegated tulips to bulbs of uniformly coloured tulips . It tookNarcissus pseudonarcissus) flowers also colour break. In daffodil, the colour break is due to changes in the levels of lipid-soluble pigments called carotenoids which, in contrast to anthocyanins, accumulate in the chromoplasts of cells. In most varieties of N. pseudonarcissus colour break is rare, usually occurring only in plants displaying leaf damage and greatly reduced vigour. Viruses are likely the cause of the colour break in these flowers. For example, 74% of 'Minister Talma' daffodils naturally infected with Narcissus yellow stripe (NYSV) and Tobacco rattle virus (TRV) were found to have white blotches on their perianths [Narcissus late season yellows virus (NLSYV), Narcissus degeneration virus (NDV) and Narcissus symptomless virus (NSV) were found in two daffodils showing flower distortion, flower colour break and leaf mottling [Daffodil  and mechanical transmission tests and detected Narcissus mosaic virus (NMV), Cucumber mosaic virus (CMV), Narcissus latent virus (NLV), Narcissus tip necrosis virus (NTNV) and Narcissus yellow stripe virus (NYSV) at high incidence in all five sites examined. Many of these viruses were present in the daffodils as mixed infections, yet the researchers reported that they did not observe colour break. Similarly, tepals of 'Dutch Master' flowers that were infected with at least three different types of viruses  showed no signs of colour break (D. A. Hunter unpublished data).The rarity of colour break in nt types ,10. ClarN. pseudonarcissus prone to colour break. This type of flower was first noted in the 1920s and is characterised by opening yellow but having its corona fade to white with age. In contrast with other daffodils, the colour breaking of the reverse bicolour flower occurs in what appear to be otherwise healthy plants. Because of this, debate remains as to whether viruses are the cause. In this study we have investigated whether viral infection is linked to the carotenoid-based colour break in reverse bicolour daffodil tepals.Reverse bicolour flowers are a particular type of The occurrence of colour break was rare at the sampling sites examined and most common in the reverse bicolour type. Colour break was characterised by the presence of irregular white patches where normally yellow pigment would have been present Figure . Colour G. globosa leaves 10 days post inoculation . Local lesions on Nicotiana tabacum and Chenopodium amaranticolor were also observed after their inoculation with extracts from colour-broken flowers of 'Twilight Zone' and 'Twelve Gauge' respectively, however symptoms on G. globosa presented the most consistent relationship between the presence and absence of colour break on an indicator host.Indicator plants were initially used to test for viral presence in 10 colour-broken reverse-bicolour flowers collected from three different locations in New Zealand . Tepal extracts from all 10 colour-broken flowers caused formation of red ringspot lesions in n Figure , Table 1Dot immunobinding assay-enzyme linked immunosorbent assay (DIBA-ELISA) using a broad-spectrum anti-potyviral monoclonal antibody showed that there was no correlation between the presence of potyviruses in the tepals and colour break Table . PotyvirTransmission electron microscopy (TEM) was used to examine the quantity and morphological characteristics of the viral particles present in a selection of the colour-broken and non-colour-broken flowers used in the indicator plant analysis. The selection encompassed all three geographic locations. TEM revealed that the tepals of all six flowers displaying colour break contained many  rod-shaped viral particles of length 502-580 nm Figure , Table 2Viral counts were also measured in the leaf and bulb extracts of seedling 21/92A, whose colour-broken tepals had the highest viral counts. In 21/92A, viral counts were found to be much lower in the vegetative tissues compared with the tepal tissues . Electrophoresis of the putative viral sequences amplified from the tepal cDNA by reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers to potex-, poty-, and carla-viral family members showed that colour break correlated strongly with the presence of viral RNA Figure .AY225449 and Dutch strain, accession D13747)  Table  also proATATTCTTGTGAGCAGCC-5330 in the 26 K protein of NMV (bases in NMV genomic sequence in common with the primer are in bold and underlined) and binding of the oligo(dT)-based primer (KSDT) to the end of the sequence (position 6956). The potyvirus NLSYV was also identified in one of the sequencing reactions  were associated with colour break in the reverse bicolour daffodils. To give the best chance of detecting as many different types of viruses as possible, the procedure was carried out at a low annealing temperature with degenerate primers shown previously to amplify successfully members of the potex-, poty- and carlaviral families ,12. The G. globosa after inoculation with colour-broken, but not non-colour-broken flowers is characteristic of NMV infection [All of the independent tests carried out pointed to NMV being the virus most closely associated with colour break in the reverse bicolour daffodils. For instance, the red ringspot lesions seen in nfection , and thenfection ,13. Howepersonal communication).NMV was first described and in detail by Brunt . The virNarcissus spp. for viral infection in the Otago Province of New Zealand using ELISA and mechanical transmission tests. A high incidence of viral infection was found in the five sites examined. Indirect ELISA showed that NMV was one of the five viruses detected and that it was present at all five sites in single or mixed infections. They reported that plants infected only with NMV (based on their detection methods) showed mild to conspicuous leaf mosaic symptoms and reported that no colour break symptoms were observed. The absence of colour break among the daffodils surveyed by Clark and Guy [In a separate study, Clark and Guy  surveyed and Guy  is presuunpublished information)" support our findings that NMV infection is closely linked to colour break in reverse bicolours as both of these cultivars are of that type . However, to demonstrate causality will require inoculation experiments. We also cannot rule out that it could be a viral titre phenomenon and not specific to a particular virus.Unpublished data by Brunt  that "soThe ubiquitous occurrence of NMV in daffodil cultivars is intriguing given no vector for transmission has yet been discovered and direct spread by mechanical transmission is slow. Brunt  reportedThe widespread occurrence of NMV and the inconspicuous symptoms it produces in many daffodil cultivars makes it difficult for the grower to prevent colour break from occurring as it appears that there is a 'silent' reservoir of the virus which can serve as inocula for infection of reverse bicolours. Because of this, growing this type of flower away from other cultivars may be the best way of preventing their colour break.The findings of this study and the previous research have demonstrated that different viruses are responsible for colour break problems in different ornamentals or even different cultivars of the same species. However, little is known about the mechanism of colour break caused by viruses at the cellular level and why in daffodils the reverse-bicolour flowers seem particularly prone to colour break and why NMV seems particularly linked to the process. It is of particular interest that distinct pigment biosynthetic pathways are affected in response to viral infection in tulips (vacuolar-located anthocyanins) and daffodils (plastid-located carotenoids). More study is thus required to understand the molecular basis of viral-induced colour break in flowers.Daffodil material was sourced from three geographic locations in New Zealand; two in the North Island  and one in the South Island (Nelson). Cut flowers or whole plants were transferred directly to the laboratory from the two nearby locations (Ashhurst and Foxton) and via overnight courier from the distant location (Nelson). Experimental materials were stored either at -20°C or 4°C until needed.Chenopodium amaranticolor, Gomphrena globosa, Phaseolus vulgaris and Nicotiana tabacum) dusted with carborundum abrasive. The plants were grown at 18-22°C and monitored over 3 weeks for the development of symptoms. Leaf tissues for DIBA-ELISA were crushed and diluted 1:20 in 0.05 M Tris buffer pH 7.5 + 0.2 M NaCl. Duplicate 20 μL drops were spotted on Zeta Probe blotting membrane  nitrocellulose paper and processed according to Hammond & Jordan [Excised daffodil tepal tissues were wrapped in foil and dried in a glass chamber at 4°C in the presence of silica gel and transported overnight in a courier bag from The New Zealand Institute for Plant & Food Research Limited (PFR), Palmerston North site, to the Christchurch PFR site. For viral transmission tests, ~0.1 g tepal tissues were ground in phosphate buffer (pH 7.5) + 0.05% bentonite (modified Yarwood's buffer) and rubbed onto leaves of indicator plants  and negatively stained with 2% uranyl acetate and observed with a Philips 201C TEM. Particle counts were obtained from the photographs taken from at least three randomly chosen fields per TEM grid, and expressed as viral counts per view at magnification of 21200 x.To observe virus particles, daffodil tepal leaf and bulb tissues were crushed in the same way in a plastic pocket and the slurry then centrifuged at 10000 et al. [RNA was isolated from daffodil tepals that were frozen in liquid nitrogen and ground in a mortar with a pestle using a modified Wan and Wilkins hot borate method  describeet al. . First set al. ,12. The http://www.ncbi.nlm.nih.gov. Conditions for the virus-specific PCR consisted of an initial denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 30 s/60°C for 30 s/72°C for 30 s and final extension at 72°C for 5 min. The amplified products were TA-cloned into the pGEM®-T Easy vector system II . Sequencing was carried out as a commercial service by the DNA Sequencing Facility, University of Waikato, Hamilton, NZ. The identity of the amplified products was confirmed by sequence comparison in GenBank using the Basic Local Alignment Search (BLASTN) algorithm [http://www.ncbi.nlm.nih.gov.The primers used for virus-specific PCR to generate short amplicons of NMV and NLSYV were designed using the PrimerSelect module of Lasergene7 software  as short amplicons have been shown to increase the sensitivity and accuracy of PCR . The virlgorithm  and 'OthThe authors declare that they have no competing interests.DH conceived and organised the project, did the RT-PCR analysis and wrote the manuscript. JF did the indicator plant and DIBA-ELISA analysis. KD contributed to experimental design and writing of the manuscript. HZ contributed to experimental design, did the TEM work and contributed to writing of the manuscript. All the authors have read and approved the final manuscript.


In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing and mitigating future outbreaks.Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10 In Bangladesh, outbreaks of Nipah virus occur almost every year, resulting in respiratory and neurological disease with high case-fatality rates. Based on epidemiological data Nipah virus is thought to be transmitted from fruit bats to humans via drinking of date palm sap contaminated by bats that drink from the sap stream or collection vessel during collection. Additionally, human-to-human transmission has been shown to occur. Here, we experimentally modeled the proposed transmission cycle of Nipah virus in Bangladesh in Syrian hamsters. Hamsters that drank artificial palm sap containing high doses of Nipah virus became infected with the virus and developed neurological signs of disease. Virus replication occurred mainly in the respiratory rather than the intestinal tract. Most importantly, hamsters infected with Nipah virus through drinking of contaminated palm sap could transmit the virus to uninfected cage mates. As treatments for Nipah virus are currently unavailable and medical interventions are difficult to implement in rural outbreak areas, our best hope to prevent or intervene in future outbreaks of Nipah virus lies in the potential to block transmission from bats to humans and from human to human. Understanding how Nipah virus is transmitted is essential to achieve this. Nipah virus first emerged in 1998 during a large outbreak of encephalitis and respiratory disease in Malaysia and Singapore, causing 276 cases of encephalitis with 106 fatalities In addition to the initial zoonotic transmission, subsequent human-to-human transmission also plays an important role in the epidemiology of Nipah virus outbreaks in Bangladesh. It was estimated for the outbreaks in Bangladesh between 2001 and 2007 that approximately 50% of cases was the result of human-to-human transmission Thus far, the epidemiological association between drinking date palm sap and Nipah virus infection has not been confirmed experimentally. Therefore, we set out to assess the ability of date palm sap to function as a vehicle for zoonotic transmission of Nipah virus using a well-established small animal model for Nipah virus pathogenesis and transmission, the Syrian hamster 3, 105 and 107 TCID50/ml) of Nipah virus (strain Bangladesh/200401066) in artificial palm sap was determined at 22°C. Of note, between October and March  the average temperature in Bangladesh fluctuates between 20°C and 28°C. No significant difference was detected between the slopes of the three lines . In addition, the slopes of the three groups were also not significantly different from zero . Thus, no significant reduction in Nipah virus infectious titer was observed at any of the dilutions for at least 8 days at 22°C . Eight hamsters were monitored daily for signs of disease for up to 28 days post inoculation (dpi). One hamster succumbed to respiratory disease on 6 dpi; a second hamster was euthanized on 12 dpi , likely indicating an active infection. In comparison, all intranasally inoculated hamsters shed virus from the nose for up to 7 days and from the throat up to 11 dpi .8 TCID50 of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. Nasal, oropharyngeal, urogenital and rectal swabs were collected up to 11 days after supplying the hamsters with artificial palm sap. All eight tested hamsters shed virus for several days, mainly via the oropharynx and, at later time points, the intestinal tract  and singly housed to examine transmission via fomites, direct contact or aerosols as described previously We have recently shown that Nipah virus  is transmitted between Syrian hamsters primarily through direct contact 8 TCID50 of Nipah virus (strain Bangladesh/200401066). After two days, when the hamsters had drunk the artificial palm sap, drinking bottles were replaced with bottles containing water and a naïve hamster was added to each cage. At 28 dpi the naïve hamsters were euthanized and sera were collected. Out of 24 naïve hamsters, 2 hamsters showed presence of antibodies directed against Nipah virus in ELISA, likely indicating that Nipah virus was transmitted to these hamsters.Next, we set out to determine if hamsters infected with Nipah virus through drinking of artificial palm sap containing Nipah virus could transmit the virus to naïve hamsters via direct contact. Of note, a larger number of animals was used in this experiment since the hamsters infected with Nipah virus through drinking of artificial palm sap shed a lower amount of virus than intranasally inoculated hamsters  and tranEpidemiological investigations in Bangladesh suggest that Nipah virus is introduced into the human population via the consumption of raw date palm sap. This study provides the first experimental evidence for the transmission of Nipah virus via the consumption of palm sap containing Nipah virus, resulting in neurological signs of disease in Syrian hamsters. Although these findings do not directly demonstrate that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide experimental support for the current hypothesis, based on epidemiological observations, of the zoonotic introduction of Nipah virus via contaminated date palm sap.Nipah virus was very stable in artificial palm sap, likely due to its neutral pH and high sugar content. Nipah virus was preserved much better in artificial palm sap than on the surface of fruit or in fruit juice Based on virus distribution in tissues of infected hamsters and the presence of vRNA mainly in throat swabs rather than urogenital or rectal swabs, the porte d'entrée for the initial Nipah virus infection upon drinking of artificial palm sap containing Nipah virus was the respiratory tract rather than the intestinal tract. This finding was further strengthened using fluorescently labeled Nipah virus to visualize the deposition of virus upon inoculation via the nose, gavage or upon drinking. These experiments clearly showed that during drinking the virus does not only end up in the intestinal tract but some of the volume is also deposited in the lungs, thereby explaining the replication of virus in respiratory tissues. However, the main disease manifestation in hamsters infected through drinking was the development of neurological signs, suggesting that the animals became infected with a relatively low dose, despite the high amount of virus present in the artificial palm sap. Previous studies have shown that inoculation of hamsters with a low dose of Nipah virus results in neurological signs of disease, whereas a high dose results in respiratory disease In one of the hamsters infected with Nipah virus through drinking of palm sap, virus was detected by immunohistochemistry in the olfactory bulb, indicating that virus traveled from olfactory neurons in the nasal turbinates through the cribriform plate into the olfactory bulb and from there further into the central nervous system. Although this route of Nipah virus into the brain has been described before Besides the ability of Nipah virus to infect hamsters via the drinking route, we showed here that Syrian hamsters infected with Nipah virus through drinking of palm sap containing Nipah virus can transmit the virus through direct contact with naïve hamsters. Transmission upon drinking of Nipah virus-containing artificial palm sap was less efficient than upon intranasal inoculation with Nipah (strain Bangladesh/200401066), likely as a result of decreased virus shedding upon infection through drinking. Within the transmission model for Nipah virus in Syrian hamsters no differences were observed in transmission route or efficiency between the Malaysian Prophylactic or therapeutic intervention measures are currently not available to prevent, treat or contain zoonotic transmission of Nipah virus. Moreover, medical interventions might be difficult to implement in rural outbreak areas. Therefore, our best hope to prevent or intervene in future outbreaks of Nipah virus lies in the potential to efficiently block zoonotic and human-to-human transmission and thereby spread of the outbreak. Currently, efforts are underway in Bangladesh to prevent zoonotic transmission of Nipah virus from fruit bats to people by restricting access of bats to date palm collection pots and thereby preventing contamination of the date palm sap with Nipah virus All animal experiments were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories, and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in an AAALAC-approved facility.Nipah virus (strain Bangladesh/200401066) was kindly provided by the Special Pathogens Branch of the Centers for Disease Control and Prevention, Atlanta, Georgia, United States. This strain (SPBLOG# 200401066) was isolated from a throat swab collected from patient #3001 on January 22 2004 in Bangladesh. This patient was a 10-year old male who developed altered mental status on January 21 and cough and breathing difficulties later that day. The patient was admitted to Goalando Hospital, Bangladesh, on January 22. None of the patient's contacts developed Nipah virus infection; the patient is presumed to have been infected via direct spillover from the bat reservoir . The virus isolate was propagated in Vero C1008 cells in DMEM (Sigma) supplemented with 10% fetal calf serum, 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco). For fluorescent labelling of virus, Nipah virus-containing cell supernatant was cleared by low speed centrifugation and virus was pelleted by spinning 2 hours at 21000 rpm in the ultracentrifuge. The pellet was resuspended in 1 ml PBS and loaded onto a 20%–60% (w/w) sucrose gradient and centrifuged overnight at 39000 rpm. The virus fraction was collected and pelleted once again by centrifuging 2 hours at 21000 rpm; the pellet was resuspended in 1 ml PBS. Purified Nipah virus particles were labeled using an Alexa Fluor 680 Protein Labeling Kit (Molecular Probes). Excess dye was removed by dialyzing against PBS.Artificial palm sap was prepared based on a literature report 7 TCID50/ml, aliquotted into 1 ml aliquots and incubated at 70°C or 100°C for up to one hour.Nipah virus (strain Bangladesh/200401066) was added to artificial palm sap at the desired concentration, aliquotted into 1 ml aliquots and incubated at 22°C or 28°C for up to eight days. The stability data were analyzed using the linear regression model in the GraphPad prism 6 software package. For inactivation of Nipah virus in artificial palm sap, Nipah virus (strain Bangladesh/200401066) was added to artificial palm sap at 10tm:AURA, Harlan Laboratories) were inoculated with Nipah virus via different routes. One group received 107 TCID50 Nipah virus (strain Bangladesh/200401066) via intranasal inoculation in a total volume of 80 µl. One group received 107 TCID50 Nipah virus (strain Bangladesh) via gavage in a total volume of 500 µl. The remaining two groups received 107 and 5×108 TCID50 Nipah virus (strain Bangladesh/200401066), respectively through drinking of artificial palm sap. Animals were housed singly and supplied with 30 ml artificial palm sap containing a total dose of 107 or 5×108 TCID50 Nipah virus instead of drinking water. When animals had drunk all artificial palm sap, in about 2 days, they were again supplied with drinking water. Nasal, oral, urogenital and rectal swabs were collected daily from eight hamsters inoculated via all four different routes. Swabs were collected in vials containing 1 ml DMEM supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin. On days 2, 4, 8, 12 and 28 post inoculation 8 animals from each inoculation group were euthanized and blood, trachea, lungs, heart, liver, spleen, kidney, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon , bladder, brain and nasal turbinates were collected for virological  and histopathological  analysis. Hamsters used for histopathological analysis were anaesthetized using ketamine (80–100 mg/kg) and xylazine (7–10 mg/kg) and perfused with PBS containing 5 mM EDTA, followed by 4% paraformaldehyde. Tissues of interest were then further fixed according to BSL4 standard operating procedures for a minimum of 7 days in 10% neutral buffered formalin.Four groups of 40 6–8 week old female Syrian hamsters (HsdHan7 TCID50 of fluorescently labeled Nipah virus (strain Bangladesh/200401066). To prevent fusion of virus particles with target cells, hamsters were euthanized ten minutes after inoculation and the respiratory tract was excised; ex vivo imaging was subsequently performed on the head and respiratory tract in an IVIS Spectrum imager (PerkinElmer). Images were acquired using an excitation wavelength of 675 nm with an emission scan at 720, 740, 760 and 780 nm at field of view B (6.6 cm) in auto-exposure mode with medium binning, f-stop 3. Following acquisition, images were unmixed in Living Image 4.2 with tissue autofluorescence subtracted. The resulting AF680 image was used for subsequent analysis. Rectangular Regions of Interest were drawn around the entire lung, trachea or nasal tract. The resulting average radiant efficiency was used to determine the quantity of labelled virus that was detected in the respiratory tract (combined trachea and lungs) or in the nasal tract (head).To visualize the deposition of virus in the hamster respiratory tract after inoculation, 3 hamsters per inoculation route were inoculated intranasally, esophageally and via drinking as described above with 107 TCID50 of Nipah virus (strain Bangladesh) in a total volume of 80 µl. On day 4 post inoculation, hamsters were euthanized and a single naïve hamster was placed in each cage. For direct contact transmission experiments, eight 6–8 week old female singly housed Syrian hamsters were inoculated intranasally with 107 TCID50 of Nipah virus (strain Bangladesh/200401066) in a total volume of 80 µl. On day 1 post inoculation, a naïve hamster was added to each cage. For aerosol transmission experiments, eight 6–8 week old female Syrian hamsters were inoculated intranasally with 107 TCID50 of Nipah virus (strain Bangladesh/200401066) in a total volume of 80 µl and singly housed in specially designed aerosol transmission cages. On 1 dpi, a naïve hamster was placed on the opposite side of the inoculated hamster. The hamsters were separated by two stainless steel grids, allowing airflow from the inoculated to the naive hamster but preventing direct contact and fomite transmission. In all transmission experiments, nasal and oropharyngeal swabs were obtained from inoculated and naïve hamsters daily and the bodyweight of naïve hamsters was determined. Upon signs of severe disease, inoculated and naïve hamsters were euthanized; remaining hamsters were euthanized four weeks post exposure.To determine the transmission route for Nipah virus (strain Bangladesh/200401066) we used the recently described Syrian hamster transmission model 8 TCID50 of Nipah virus (strain Bangladesh/200401066) instead of drinking water. When animals had drunk all artificial palm sap they were again supplied with drinking water and a naïve hamster was added to each cage. Nasal and oropharyngeal swabs were obtained from inoculated and naïve hamsters daily and bodyweight of naïve hamsters was determined. On signs of severe disease, inoculated and naïve hamsters were euthanized; remaining hamsters were euthanized four weeks post exposure.To determine whether Nipah virus (strain Bangladesh/200401066) is transmitted via direct contact after infection through palm sap containing Nipah virus, 24 6–8 week old female singly housed Syrian hamsters were supplied with 30 ml artificial palm sap containing 5×1050 was calculated from 5 replicates by the method of Spearman-Karber. Tissue homogenates were prepared by adding 1 ml DMEM to the weighed tissue and homogenizing using a TissueLyzer II (Qiagen). Homogenates were centrifuged to clear the homogenate before inoculating cells.Virus titrations were performed by end-point titration in VeroC1008 cells. VeroC1008 cells were inoculated with tenfold serial dilutions of swab medium or tissue homogenates. One hour after inoculation, the inoculum was removed and replaced with 200 µl DMEM supplemented with 10% fetal calf serum, 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco). Five days after inoculation, cytopathic effect (CPE) was scored and the TCIDHistopathology and immunohistochemistry was performed on hamster tissues. Necropsies and tissue sampling were performed according to a standard protocol approved by the Institutional Biosafety Committee. After fixation for 7 days in 10% neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (H&E) and an immunohistochemical method using a rabbit polyclonal antiserum against the Nipah virus nucleoprotein 50 equivalents in the samples.RNA was extracted from swab samples using the NucleoSpin 96 Virus Core kit (Macherey-Nagel) and a Corbett Robotics model CAS 1820 automatic RNA extractor. RNA was eluted in 100 µl. 5 µl RNA was used in a one-step real-time RT-PCR targeted at the NP gene using the Rotor-Gene probe kit (Qiagen) according to instructions of the manufacturer (primer and probe sequences are available on request). In each run, standard dilutions of a titered virus stock were run in parallel, to calculate TCIDAntibody responses were measured in an enzyme-linked immunosorbent assay (ELISA) using inactivated Nipah virus  as the antigen. Nipah virus-containing cell culture supernatant was concentrated and purified by centrifuging for two hours at 21000 rpm over a 20% sucrose cushion. The pellet was resuspended in PBS and Triton X-100 was added to a final concentration of 1%; the preparation was then inactivated with γ-radiation according to standard operating procedures. This suspension was used to coat immuno 96 microwell maxisorp plates (NUNC) at 4°C overnight. Subsequently, plates were blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1.5 hours at 4°C. After 3 washes with PBST, 50 µL of diluted serum samples were added, and the plates were incubated for 1 hour at 37°C. Bound antibodies were detected after 3 washes using an anti-hamster secondary antibody conjugated with horseradish peroxidase . Following incubation for 1 hour at 37°C, bound HRP was detected using the ABTS Peroxidase Substrate System (KPL). The absorbance at 405 nm was measured using a microplate spectrophotometer. Sera were considered positive when absorbance was higher than three standard deviations above the mean of negative control sera.Figure S17 (black triangles) and 105 (black squares) TCID50/ml in artificial palm sap and left at 28°C for 8 days. Samples were taken at the indicated time points and virus titer in those samples was determined by titration on Vero C1008 cells. The stability data were analyzed using the linear regression model in the GraphPad prism 6 software package. The regression line (solid) is plotted together with the 95% confidence interval (dotted line). (B) Nipah virus (strain Bangladesh/200401066) was diluted to 107 TCID50/ml in artificial palm sap and left at 70°C (green line) or 100°C (blue line) for 1 hour. Samples were taken at the indicated time points and virus titer in those samples was determined by titration on Vero C1008 cells. Geometric mean titers were calculated from three replicates; error bars indicate standard deviation; dotted line indicates the cut off value of the assay.Stability of Nipah virus in artificial palm sap. (A) Nipah virus (strain Bangladesh/200401066) was diluted to 10(TIF)Click here for additional data file.Table S11 Viremia was determined by real-time RT-PCR.Tissue distribution of Nipah virus in hamsters inoculated intranasally, esophageally or via drinking as determined by virus titration. Numbers in the table indicate number of animals in which virus was detected in the indicated tissues at the indicated time points; total number of animals is indicated at the top of the column. (DOCX)Click here for additional data file.Table S21 Tissues were collected when animals were euthanized due to severity of disease signs. 2 nc: not collected.Tissue distribution of Nipah virus in hamsters inoculated intranasally, esophageally or via drinking as determined by immunohistochemistry. Numbers in the table indicate number of animals in which viral antigen was detected in the indicated tissues at the indicated time points; total number of animals is indicated at the top of the column. (DOCX)Click here for additional data file.


Ultrastructural analysis of a filovirus assembling within infected eukaryotic cells reveals differences in structure and assembly mechanisms between related RNA viruses. When it comes to nasty pathogens, Marburg virus is among the nastiest. Cousin to Ebola virus, Marburg causes fever, rash, delirium, and severe hemorrhaging, often ending in organ failure and death. It is rare in the wild, but was a central focus of weaponization by the Soviet Union, and remains a concern for terrorism experts who fear its lethal potential and resistance to treatment.One reason that treatments have proved so elusive is because the virus is so hard to work with—hazmat suits, self-contained breathing gear, and electronically secured airlocks are all required for even the simplest of studies with live virus. But another reason is that the virion  is heterogeneous in shape, and that heterogeneity has confounded standard imaging techniques , which require purified, identical particles to obtain their highest resolution.PLoS Biology, Tanmay Bharat, John Briggs, and colleagues get around that problem by using a sophisticated combination of imaging techniques that, through multiple iterations, provides the first clear three-dimensional picture of the intact Marburg virion structure.In this issue of Marburg and Ebola are the only two identified viruses that make up the small family of filoviruses, a relative of the larger family of rhabdoviruses. Both families contain an RNA strand coated with proteins, but the rhabdoviruses are more structurally homogenous, which makes them easier to study. The prototypical rhabdovirus is vesicular stomatitis virus (VSV), and is the only member of the family for which detailed information is available on structure and host–cell interactions. But it has not been clear how broadly insights from VSV can be applied to related viruses.The authors began by isolating Marburg virions from infected cells, and imaging them using cryoEM, a technique that freezes samples to prevent their distortion in the evacuated atmosphere of the electron microscope. The virion occurred in three forms: filamentous, round, and a combination particle shaped like the number 6 (these three forms likely represent a continuum of possible virion shapes). In the next step, using the same principle as a CAT scanner, they collected many images taken at different angles, and processed them to create three-dimensional images of the virions.Turning their attention to the filamentous particle, they found it was composed of concentric layers of different density, which they suspected corresponded to the different proteins known to make up the virion. To determine which was located where, they sectioned the virions and stained them with antibodies to each of the known proteins in succession, then imaged them in the electron microscope. (Gold particles attached to the antibodies show up as jet black dots in the EM). Their work allowed them to assign one or more proteins to each layer, with the viral RNA linked to a protein at the center. They further confirmed their results by purifying the RNA-binding protein (nucleoprotein) alone, which could be imaged in the cryoEM.The core of the filament, called the nucleocapsid, was studded with boomerang-shaped protrusions that spiraled around the outside, composed of two different viral proteins, and an inner, more dense set of lobes, each of which contained the RNA-nucleoprotein complex. The average nucleocapsid contained over 3,000 copies of the nucleoprotein, arranged in a helix and each binding to six RNA bases of the viral genome .The viral life cycle requires nucleocapsids to bud from the host cell membrane to form the infectious particle. Using cryoEM, the authors found that the host cell's filopdia, membrane extensions from which the virus buds, could contain more than one nucleocapsid. The nucleocapsid has a directionality, with the “pointed” end always being first to be enveloped by host cell membrane, and the “barbed” end following. In contrast, VSV buds with its barbed end first. In that virus, the earliest synthesized part of the genome, the so-called three-prime end, is packed in the barbed end, while in a related virus, respiratory syncytial virus (RSV), it is in the pointed end. If Marburg virus orients its RNA as RSV does, the three-prime end of its genome would also be the first to bud, as it is in VSV.The results presented are the first detailed three-dimensional look at the structure of the Marburg virus, and thus provide a wealth of new information that will likely lead to a better understanding of the virus's lethal biology. It should also illuminate aspects of related viruses, including Ebola and rabies, which also bear single-stranded RNA genomes and have many similar structural features, but differ in some fundamental details of their life cycles.Bharat TAM, Riches JD, Kolesnikova L, Welsch S, Krähling V, et al. (2011) Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells. doi:10.1371/journal.pbio.1001196


Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze–thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition.Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR  adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in  Lipothrixviridae, Rudiviridae and Ampullaviridae or circular for the Bicaudaviridae, Fuselloviridae and Guttaviridae and some unclassified viruses, with the genome sizes ranging from 14.7 kb [SSV2 (Sulfolobus spindle-shaped virus 2)] to 75 kb [STSV1 (Sulfolobus tengchongensis spindle-shaped virus 1)]   (Table 1virus 2)  and, lesMembers of the Sulfolobales also host a family of conjugative plasmids . These pS. islandicus species have been presented previously [in silico analyses to derive either from specific viral families or the conjugative plasmid family [Sulfolobus solfataricus P2, protospacers incorporated into loci C, D, E and F carry -CCN- PAM sequences, whereas loci A and B require -TCN- sequences -based immune systems and their properties are summarized for a few widely studied, and phylogenetically diverse, species in eviously . Extendeeviously ,16, and d family –20. Protequences  where eaS. islandicus shared a crRNA (CRISPR RNA)-processing enzyme Cas6 (where Cas is CRISPR-associated) and single spacer-specific crRNAs with a coexisting subtype I-A system [Both Type I and diverse Type III systems are generally present within cells and have the potential to generate different interference complexes targeting viral or plasmid DNA or their encoded RNAs. Subtype I-A and subtype III-B systems are widespread, whereas subtype I-D, subtype III-A and other unclassified Type III systems occur less frequently, and subtype I-B systems are found rarely ,21,22. MSulfolobus strains suggests that they periodically exchange intercellularly [cas gene cassettes of the Sulfolobales often being flanked by transposable elements and toxin–antitoxin gene pairs [The presence of between one and four different variants of Type III interference systems encoded in closely related llularly . This prne pairs .Sulfolobus species infected with viruses or carrying plasmids has proven very difficult. Numerous attempts were made in our laboratory, and in others, with single viral infections with no detectable spacer acquisition [Streptococcus thermophilus [Escherichia coli [Although considerable progress has been made in characterizing the processing mechanisms of archaeal CRISPR transcripts and the structures of the different interference complexes, we still have limited insight into mechanisms of spacer uptake into CRISPR arrays. Induction of spacer acquisition in laboratory strains of uisition ,24a. Thimophilus –27 and, hia coli –30, albeLipothrixviridae and Fuselloviridae and the tailed fusiform STSV1 contain host lipids consistent with a budding mechanism for cellular extrusion which leaves the host cell intact [This apparent contradiction may reflect, at least to some degree, differences between the virus–host relationships of archaea and bacteria. Most viruses infecting the Sulfolobales are lytic, but do not often cause cell lysis, although when cell lysis does occur, viruses are expelled via pyramid-shaped structures forming in the archaeal cell membrane ,6. In col intact –33, as hl intact . Thus arl intact . TherefoS. solfataricus P2 with SMV1 (Sulfolobus monocaudavirus 1), a single-tailed fusiform virus isolated from Yellowstone National Park [de novo spacers produced resistance to the conjugative plasmid, but did not inhibit virus propagation. In these experiments, leader-proximal regions, and the whole loci for locus A (102 spacers), locus B (94 spacers), locus C (31 spacers), locus D (95 spacers), locus E (seven spacers) and locus F (88 spacers), were screened for spacer uptake by PCR amplification over a 70 day period p.i. (post-infection). Spacer uptake adjacent to the leaders was observed exclusively for loci C and D, and, exceptionally, it occurred throughout the smaller locus E via an altered insertion mechanism [The first successful archaeal spacer acquisition was observed on infecting nal Park ,24a  [de novo spacers within a CRISPR locus of a single clone [Most laboratory studies on the mechanisms of spacer acquisition have been performed on the genetically manipulated subtype I-E system of  repeat) –30,42. Mle clone . Additiole clone . Moreovele clone .Sulfolobus subtype I-A acquisition differs in several basic respects from the above. First, Cas4 has been strongly implicated in the acquisition stage and it has been shown to generate 5′-overhangs on DNA fragments compatible with a spacer uptake mechanism [I and subtype I-AII subsystems respectively [de novo pMGB1 spacers in either locus C or locus D showed that only 50% (25) were unidirectional. Finally, two apparently different spacer addition mechanisms have been characterized exclusively for Sulfolobus. Thus specific addition occurred at the leader-adjacent repeat as in S. thermophilus and E. coli and, in addition, at repeats located throughout CRISPR locus E of S. solfataricus P2. However, it remains to be demonstrated how commonly the latter mechanism operates. At present, there is no evidence for spacer-acquisition priming in Sulfolobus, although we cannot eliminate the possibility that the multiple host spacers matching SMV1 activate spacer acquisition which is then directed exclusively to pMGB1. There is also currently no direct evidence for coupling between the interference and spacer-acquisition reactions in Sulfolobales organisms.echanism ,40. Possectively . Thirdlyin vivo in the crenarchaeon Pyrococcus furiosus and to target crRNA-complementary RNAs in vitro for Sulfolobus and, potentially, they can target and degrade any mRNA, non-coding RNA or antisense CRISPR RNA [S. islandicus can co-function with a CRISPR locus and a Cas6-processing enzyme associated with a subtype I-A system [We still know relatively little about the disparate Type III CRISPR systems. They generally occur in multiple diverse copies per cell  and theiISPR RNA ,40. TheyA system . CurrentA system .de novo spacer matches to SMV1 were identified [de novo pMGB1 spacers matching this element, we found that they all matched the viral element to some degree, with 12 showing perfect matches. The S. solfataricus P2 host also carries copies of closely related IS elements, but only one nearly identical sequence was found carrying a single mismatch  and seven in locus D  and each exhibiting cognate PAM sequences [de novo spacers matching the IS element would be unlikely to inhibit virus propagation. pMGB1 also carries two non-identical copies of the SMN2 MITE (miniature inverted repeat transposable element)-like element (In the previous study, working with partially assembled genomes of the SMV1 virus and pMGB1, no entified ,24a. Howentified , that wamismatch . The IS  element  which shDirect evidence was found for transpositional activity occurring during spacer acquisition. pMGB1 carries two dissimilar mobile OrfB elements , of whicS. solfataricus P2 during the interference stage. CRISPR systems were challenged by transformed plasmids carrying a protospacer sequence matching a specific CRISPR spacer and, in some surviving transformants which carried plasmids maintained under selection, the matching spacer had been specifically targeted by an IS element which destroyed the sequence match to the plasmid [Interdependence of a CRISPR immune response and transpositional activity was also demonstrated earlier for  plasmid .S. solfataricus P2, and we finally succeeded with a mixture of the tailed fusiform virus SMV1 and conjugative plasmid pMGB1 [Clearly, we are still in the early stages of understanding the mechanisms involved in spacer acquisition and maintenance in CRISPR loci and, as indicated above, there are significant differences in the molecular mechanisms of spacer acquisition operating for different types of CRISPR system and in phylogenetically diverse archaea and bacteria. All of our experiments with diverse single archaeal viral infections failed to initiate spacer acquisition in id pMGB1 ,24a. SurSulfolobus [S. solfataricus strains isolated at different times over a 30-year period which show extensive sequence conservation of their CRISPR loci with very few de novo spacers added [Sulfolobus species.The hyperactive uptake of immensely diverse spacers observed in the laboratory for a subtype I-A system of lfolobus ,24a, andlfolobus ,29,41, irs added ,18,23, c