Cryptococcus gattii is endemic in various parts of the world, affecting mostly immunocompetent patients. A national surveillance study of cryptococcosis, including demographical, clinical and microbiological data, has been ongoing since 1997 in Colombia, to provide insights into the epidemiology of this mycosis.Cryptococcosis due to C. gattii were reported . Norte de Santander had the highest incidence (0.81 cases/million/year), representing 33.3% of all cases. The male: female ratio was 3.3∶1. Mean age at diagnosis was 41±16 years. No specific risk factors were identified in 91.1% of patients. HIV infection was reported in 6.7% of patients, autoimmune disease and steroids use in 2.2%. Clinical features included headache (80.5%), nausea/vomiting (56.1%) and neurological derangements (48.8%). Chest radiographs were taken in 21 (46.7%) cases, with abnormal findings in 7 (33.3%). Cranial CT scans were obtained in 15 (33.3%) cases, with abnormalities detected in 10 (66.7%). Treatment was well documented in 30 cases, with most receiving amphotericin B. Direct sample examination was positive in 97.7% cases. Antigen detection was positive for all CSF specimens and for 75% of serum samples. C. gattii was recovered from CSF (93.3%) and respiratory specimens (6.6%). Serotype was determined in 42 isolates; 36 isolates were serotype B (85.7%), while 6 were C (14.3%). The breakdowns of molecular types were VGII (55.6%), VGIII (31.1%) and VGI (13.3%). Among 44 strains, 16 MLST sequence types (ST) were identified, 11 of them newly reported.From 1,209 surveys analyzed between 1997–2011, 45 cases caused by C. gattii has a low prevalence in Colombia, with the exception of Norte de Santander. The predominance of molecular type VGII is of concern considering its association with high virulence and the potential to evolve into outbreaks.The results of this passive surveillance study demonstrate that cryptococcosis caused by  Cryptococcus neoformans and C. gattii, with the most serious manifestation of disease being infection of the central nervous system (CNS). C. neoformans tends to cause disease in immunosuppressed patients, especially those infected with HIV, while C. gattii affects immunocompetent patients preferentially. C. gattii is usually endemic in tropical and subtropical areas. However, highly virulent strains have recently emerged in temperate areas, such as British Columbia in Canada and the Pacific Northwest of the United States. The Colombian national cryptococcal survey characterized the demographic, clinical manifestations and microbiological aspects of C. gattii cryptococcosis. An annual average incidence of infection of 0.07 cases/million inhabitants/year was determined. In contrast, in Norte de Santander the incidence reached 0.81 cases/million inhabitants/year. The national prevalence was 3.7% among all forms of cryptococcosis. Involvement of the CNS (88%) was the commonest clinical manifestation of cryptococcosis. Molecular type VGII, which is the same molecular type as described in the recent outbreaks of this mycosis, was the most prevalent. Overall, clinical C. gattii strains from Colombia showed great genetic diversity. This work contributes to knowledge of the global epidemiology of cryptococcosis and its clinical behavior in Colombian patients.Cryptococcosis is caused by  Cryptococcus neoformans and Cryptococcus gattiiC. gattii has been recognized as a distinct species from C. neoformans due to differences in the morphology of the basidia, environmental niches, multiple gene genealogies, unique patterns generated by different molecular typing techniques, inefficient crossing of species with the production of sterile progeny and a lack of genetic recombination C. gattii can be easily and reliably differentiated from C. neoformans through a simple phenotypic procedure, growth on CGB  culture medium C. gattii assimilates glycine, is resistant to canavanine and changes the color of the media due to an alteration in pH when creatinine is degraded into ammonia. C. neoformans is not able to assimilate glycine; therefore, it does not grow on this media Cryptococcosis is a fungal disease that affects humans and animals and is caused by two species, C. neoformans is responsible for most cases of cryptococcosis worldwide C. gattii was considered rare, however cryptococcosis by C. gattii has gained importance because of its emergence in 1999, which resulted in an outbreak on Vancouver Island and other closely related regions in British Columbia, Canada The two species cause different clinical manifestations and have different biological characteristics C. gattii was restricted to tropical and subtropical regions Previously, it was thought that C. gattii is recent, although the first publications of meningeal cryptococcosis was in a child from the Congo in 1970 The knowledge about the epidemiology of C. gattii is a fungal pathogen globally set, with a potential primary ecological niche being associated in some way with decaying wood from a large range of tree species C. gattii has been associated with at least 54 species of trees native to tropical, subtropical and temperate regions C. gattii  are accepted according to the characterization by PCR fingerprinting, Random Amplification of Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), MLMT and MLST analyses, which are different to the genotypes of C. neoformans (VNI to VNIV) C. neoformans var. neoformans x C. gattii DB, C. neoformans var. grubii x C. gattii AB) and intra-species (C. neoformans var. grubii x C. neoformans var. neoformans AD) hybrids, have been identified C. gattii received through the survey during the period of 1997–2011. Our overarching aim was to provide a broad brushstroke picture of this mycosis in Colombia.In Colombia, a national survey on cryptococcosis is ongoing since 1997, led by the Instituto Nacional de Salud in Bogotá (INS) and the Corporación para Investigaciones Biológicas in Medellin (CIB) C. gattii in Colombia, identified through an ongoing national survey.This is a descriptive study of the clinical, epidemiologic and microbiological characteristics of cases of cryptococcosis due to The information was obtained by a survey designed according to the guidelines of the European Confederation of Medical Mycology, and processed by health professionals in different public and private health care institutions in Colombia, which also sent the corresponding strains from each case to the INS for centralized genotyping.The survey contained the following information: year of diagnosis of cryptococcosis, patient's demographic data, such as gender and age, department  and place of residence, risk factors for cryptococcosis . In cases associated with HIV infection, we asked if cryptococcosis defined AIDS; the date of diagnosis of HIV, the clinical manifestations of cryptococcosis and the type of initial treatment. Also, diagnostic tests performed viz. direct examinations, cultures and the determination of the capsular antigen in serum and cerebrospinal fluid (CSF) and the findings of diagnostic imaging . In addition to the aforementioned information, the evolution of the disease was determined for patients from Norte de Santander.The study was approved by the Ethics Committee of the CIB. Additionally, it had technical and ethical approval of the INS.With the information received, a database was created using Biolomics ver. 7.5.44 , while the data were analyzed statistically using Epiinfo ver. 6.1 .Cryptococcus spp from a normally sterile site, from sputum, bronchoalveolar lavage or biopsy.A case was considered probable when there were clinical findings consistent with cryptococcosis. Cases were confirmed after isolation of a The mean annual incidence rate was determined using as denominator the Colombian population census done in 2003, an intermediate year of the surveillance, determined by DANE Strain identification was done using conventional mycology techniques. Determination of species was performed by culturing the strains on CGB media Candida albicans strain ATCC 90028, which shows an inhibition range between 32–43 mm.The susceptibility profiles to amphotericin B (AMB) using the E-test  and to fluconazole (FCZ) and voriconazole (VCZ) using disc diffusion method M44-A described by the Clinical Laboratory Standards Institute (CLSI) were determined at the CIB laboratory. Quality control was done by including the 5a or α  was determined using specific primers described previously Molecular type was determined in all strains using PCR fingerprinting with the primer (GTG)CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region, were amplified following the ISHAM consensus MLST typing scheme for C. neoformans and C. gattiiC. gattii WM 179 , WM 178 , WM 175  and WM 779 , were included in the genetic analyses for verification of the four major molecular types of C. gattiiFor 44 strains, seven unlinked genetic loci, including C. gattii, reported from January 1970 to 31 July 2014, its prevalence and the molecular types of clinical strains from the world using the following search terms in English: Cryptococcus gattii, epidemiology, meningitis, HIV, AIDS, children. Articles in Spanish and Portuguese were searched in the databases SciELO and Lilacs. Only available articles were included. In addition, some references cited in articles obtained in the primary search were included also.A search was performed using the Medline database about cryptococcosis by C. gattii.Over the period from January 1997 to December 2011, 1,209 surveys were completed, with corresponding cultured isolate, and were submitted from 76 centers in 25 departments of Colombia and Bogotá D.C. Of these, 45 (3.7%) corresponded to cases of cryptococcosis by C. gattii in Colombia was 0.07 cases per million inhabitants per year, but in Norte de Santander, it was 0.81 cases per million inhabitants per year.The number of cases received per year is shown in A preponderance of males 34 (75.5%) was found, with the male: female ratio being 3.3∶1. The average age of the patients was 40±16 years with a range of4 to 68 years. The age and gender distribution is shown in No predisposing risk factors were reported in 41 (91.1%) of patients. From the cases in which the risk factors were known, the most frequent were: HIV infection  and autoimmune disease with the use of steroids . Analysis of the Norte de Santander patients revealed that 93% had no apparent risk factor.Clinical manifestations were available for 41 patients . The mosThe most common clinical presentation was neurocryptococcosis , followed by pulmonary cryptococcosis  and disseminated disease . Clinical presentation was not determined in 1 case (2.2%).In 21 (46.7%) cases results of chest X-ray were available, with 7 (33.3%). showing abnormalities. Computed tomography (CT) of the head was conducted in 15 (33.3%) cases with abnormalities reported in 10 (66.7%).Initial antifungal therapy was reported in 30 (66.7%) surveys. AMB was used most often, in 29 (96.7%) patients.Various diagnostic methods were carried out for cryptococcal identification, including direct microscopy, determination of the capsular antigen in serum and CSF and culture of CSF and respiratory samples. The results of these tests are described in C. gattii strains. All strains were susceptible to AMB (MIC <2 µg/ml). Thirteen (31%) were susceptible (MIC ≤8 µg/ml), 14 (33.3%) susceptible dose-dependent (SDD) (MIC 16–32 µg/ml) and 15 (35.7%) resistant (MIC ≥64 µg/ml) to FCZ. With regard to VCZ, 39 (92.9%) were susceptible (MIC ≤1 µg/ml), 1 (2.4%) SDD (MIC 2 µg/ml) and 2 (4.8%) resistant (MIC ≥4 µg/ml).Susceptibility to antifungals was determined for 42/45 a (The molecular types of the 45 VG isolates in order of frequency were: VGII 25 (55.6%), VGIII 14 (31.1%), and VGI 6 (13.3%) . The disa .C. neoformans and C. gattii MLST databases accessible at http://mlst.mycologylab.org and the sequences of identified alleles were deposited in GenBank  were identified: two STs amongst the VGI strains, nine STs amongst the VGII strains  strains) and five STs amongst the VGIII strains . Eight S GenBank .D) was calculated. In general, C. gattii strains from Colombia were highly diverse (D = 0.20). The greatest diversity was identified in the molecular type VGIII (D = 0.26), followed by VGII (D = 0.48) and VGI (D = 0.56).To estimate the genetic diversity of the isolates studied, the Simpson's Diversity index  were discharged alive, while 2 (18.2%) died during hospitalization.C. gattii around the world, as well as their molecular types, when reported C. gattii globally is generally low overall The incidence of cryptococcosis caused by C. gattii infections, the value of 3.7% found in this survey reflects Colombia is a country of overall low prevalence, despite being in the torrid zone of the planet. However, Norte de Santander has a high prevalence of 33.3% (60% in patients without AIDS) C. gattii in the northern region of Brazil have been present in HIV-negative children C. gattii cryptococcosis was found in patients with AIDS, a high value for this type of population C. gattii in hospitalized AIDS patients in Botswana C. gattii was also reported from South Africa, where AIDS-related cryptococcosis due to C. neoformans is epidemic Regarding the prevalence of C. neoformans, disease referable to C. gattii is more frequent in men As in cryptococcosis patients infected with C. gattii in children domiciled in tropical areas in Brazil The presence of cryptococcosis by C. gattii has changed in the last decade, with the recognition that disease affects immunocompetent and immunosuppressed patients, including patients with AIDS, in regions of the world outside of the tropical and subtropical areas C. gattii were the use of oral steroids, the presence of pneumonia and other lung diseases C. gattii, when compared with patients infected with different strains, were significantly more likely to have predisposing conditions and respiratory symptoms and a lower probability of having CNS involvement The majority (91.1%) of the Colombian patients described in the current study did not report any apparent risk factor. There were, however, some cases in patients with AIDS or immunosuppressed by another means. The behavior of Colombian patients is in accord with the descriptions of previous studies C. gattii is clinically more virulent than C. neoformans, as well as being more likely to be a primary pathogen, with a propensity to cause multiple cryptococcal granulomas in the lungs and the brain of affected patients C. gattii from the outbreak in British Columbia induced an inflammatory response less protective because the organisms somehow inhibit the migration of neutrophils towards the sites of infection. Additionally, in contrast to C. neoformans, they fail to induce the production of protective cytokines In vitro studies of the same isolates showed that dendritic cells are able to destroy cryptococci, but C. gattii evades adaptive immunity by preventing maturation of those cells and causing an inadequate activation and proliferation of T cells C. gattii decreases the effective response of Th1/Th17 mediated by dendritic cells and regulates the low expression of lung cytokines, resulting in an inability to produce a protective immunity in immunocompetent hosts It is believed that C neoformans infections, meningoencephalitis caused by C. gattii responds more slowly to antifungal therapy, and patients require a longer duration of treatment C. gattii. In the current study a third of the patients presented with gross lung abnormalities.Compared to patient with C. gattii, pulmonary nodules or masses with diameters ranging from 5 to 52 mm in diameter and focal areas of consolidation have been described C. gattii and C. neoformans were recently revised In immunocompetent patients affected by C. gattii. The most frequently identified serotype in Colombian clinical strains was serotype B (85.7%), which is the most prevalent serotype in clinical and environmental samples. C. gattii serotype C was less common although has been associated with AIDS and with immunocompetent patients The diagnosis of the vast majority of the Colombian patients reported in the study was obtained by analysis of CSF samples. Direct microscopic examination showed high positivity (97.7%), compared with much lower rates reported for this test in patients without AIDS C. gattii were found with a clear predominance of VGII, followed by VGIII and to a lesser extent VGI. The preponderance of the molecular type VGII in Colombia is similar to that reported in Western Australia C. gattii strains because of the epidemic potential associated with the VGII molecular type.In Colombian patients, three of the four molecular types of C. neoformans and C. gattiiC. gattii strains are genetically diverse. In spite of the small number of isolates studied, several genotypes were identified belonging to the full range of molecular types, in contrast to the less diverse and rather clonal C. gattii populations reported in other countries such as Canada, USA, eastern Australia and Thailand, where few genotypes have been identified amongst a much larger number of strains The emergence of specific genotypes of endemic and epidemic disease has been reflected in the large global effort being made to increase knowledge about the population genetics of C. gattii genotypes, especially amongst the VGI isolates, for which the herein identified STs have been reported from several countries; ST51 was previously found in Australia, China, India, Mexico, Papua New Guinea and USA The finding of STs previously reported in other parts of the world, shows a wider geographic dispersion of some C. gattii are mating type aC. gattii has been recovered frequently from the environment in Colombia and in the city of Cúcuta (Norte de Santander), with both serotypes B and C having been cultured In Colombia, the majority of the strains of C. gattii to these antifungals and according with the international guidelines for countries with limited resources The initial treatment of cryptococcosis in almost all patients from Colombian was done with AMB, with or without FCZ, which is in agreement with the results of susceptibility testing of all strains of Checklist S1STROBE checklist.(DOCX)Click here for additional data file.Table S1Cryptococcus gattii cryptococcosis cases in Colombia, per state, year and molecular type. Number of cases in brackets.Number of (DOCX)Click here for additional data file.Table S2Cryptococcus gattii clinical strains.Gene Bank accession numbers for the allele types of Colombian (DOCX)Click here for additional data file.Table S3Cryptococcus gattii: Prevalence and molecular type of clinical isolates reported worldwide.(DOCX)Click here for additional data file.


Bats are natural reservoir hosts and sources of infection of several microorganisms, many of which cause severe human diseases. Because of contact between bats and other animals, including humans, the possibility exists for additional interspecies transmissions and resulting disease outbreaks. The purpose of this article is to supply an overview on the main pathogens isolated from bats that have the potential to cause disease in humans. Bats are an important reservoirs of different pathogenic agents, and many of them have already caused disease outbreaks worldwide.More than 200 viruses have been associated with bats, and almost all are RNA viruses probably owing to their great ability to adapt to changing environmental conditions through a higher genetic variability.Bacteria in bats and their putative threat to humans remain poorly studied.The understanding of the mechanisms of viral persistence in bats remains unclear.Extensive studies are needed to improve our understanding of bat–human interactions in order to design new control measures in the future.Strategies on surveillance and monitoring of disease outbreaks in bat populations need to be further developed, in particular where bats and humans are in close contact.1 Bats are essential members of the global ecosystem and humans benefit from their presence in many ways. They are involved in seed dispersal and pollination activity: tropical bats are vital – as an example – in rebuilding cut down forests and in the pollination of wild plants as bananas, avocados and dates. Furthermore, the flying mammals are the major predators of night insects, including crop and human pests.1 Finally, their guano, which is rich in nitrogen, is used as biological fertilizer.Bats, mammals of the Chiroptera order, are present all over the world with the exception of the Arctic, the Antarctic and a few oceanic islands. Bats are the only mammals with the ability to fly and are present in >1100 different species.Criminals are a superstitious cowardly lot. So my disguise must be able to strike terror into their hearts. I must be a creature of the night, black, terrible..... ‘Just then a huge bat flies in the open window’. A bat! That’s it! It’s an omen. I shall become a bat! In: Detective Comics no 33, 1939.Despite the multiple benefits attributed to these animals, since the ancient times – through myths and misapprehensions – bats have gained a bad reputation in the general public. The classical literature is full of examples in which bats are associated with evil and darkness. The roman poet Ovid narrates in the Metamorphoses that the god Bacchus transforms the daughters of Mineus, king of Boeotia, into bats as a punishment for profanating his celebration . In the 2 Bats are widespread in urban areas and come in close contact with both domestic animals and humans, contaminating houses with guano and urine, additionally, humans occasionally encroach into bat habitats.3 Their characteristic ecology undoubtedly influences the maintenance and transmission of microorganisms within the colony and directly or indirectly to humans.3Bats, however, can be involuntarily dangerous to humans. Indeed, they are natural reservoir hosts and sources of infection for several microorganisms, including pathogens that can cause severe human diseases, and are more frequently implicated in zoonotic virus emergencies.4 These intermediate hosts can be infected in many ways, including ingestion of food partially digested by bats. Frugivorous bats, in fact, cannot ingest wide amounts of food because of the aerodynamics of flight,5 therefore, they extract nutrients by chewing fruits and spitting the residues. This partially digested food dropped on the ground can then be ingested by other animals and is a potential infectious source. A similar modality of viral transmission has also been described for insectivorous bats.5 Bats can also directly infect humans.4 This can occur through ingestion of infected bat meat, as in some areas bats are a food source, or through bat’s bite as in the case of rabies virus.Microbial transmission within bat colonies is promoted by the behavior of several species of these mammals aggregating in crowded roosts. Bats can transmit infectious agents to humans through intermediate hosts, which are in close contact with humans.7Other features of bat’s life make them a good host for infectious agents. The fact that several species of bats hibernate during the winter is one of these. Although the role of the hibernation in infection dynamics has not been widely studied, it is likely that this condition can contribute to the maintenance of pathogens in the cold weather (see below: white-nose syndrome (WNS)). Moreover, unlike many other small mammals, bats live >30 years. Their long life makes them a great reservoir for pathogens and gives them many occasions to transmit them to other species. In addition, some species of bats migrate – also for distances >1000 km – allowing them to spread diseases in big areas and acquire new microorganisms.Lyssaviruses – these are apparently not pathogenic for them, suggesting that they may control viral replication more efficiently than other mammals. It has been hypothesized that there may be a relationship between flight and low virulence.8 During flight, bats show an increase in the metabolic rate and in body temperature, comparable to the fever response rendering replication of infectious agents, which are temperature sensitive, less favorable.Finally, most pathogens are not dangerous for bats and can therefore survive for long time in the host without killing it. Indeed, despite the fact that bats are infected with more different zoonotic viruses per host – with the exception of rabies virus and other In this review, we give a comprehensive overview of the microorganisms and viruses isolated from bats and their possible role in human disease.9 In fact, RNA viruses have higher mutation rates compared with DNA viruses as the viral RNA polymerases lack proofreading activity. Furthermore, RNA viruses with segmented genomes have the ability to modify their genome through genetic re-assortment . Below we report some examples of human infectious diseases associated with bat viruses.Bats are recognized as important reservoirs of different families of viruses, most of which are emerging as human pathogens, such as Ebola and Marburg viruses, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) coronaviruses. More than 200 viruses have been associated with bats, and almost all are RNA viruses probably owing to their great ability to adapt to changing environmental conditions through a higher genetic variability.Rhabdoviridae contain six genera, including Lyssavirus, the most important bat-associated virus. At least 14 species of the Lyssavirus genus can be detected in bats, which are considered the ancestral hosts for these viruses. Lyssaviruses can be found worldwide and can be classified using different criteria, such as genetic distance, antigenic patterns, geographical distribution and host range.11 The characteristic bullet-shaped virus, transmitted to humans through the bite of infected animals, causes an acute, and frequently fatal, encephalitic disease.Lyssavirus genus.12 Carini12 suggested a link between rabies infection and hematophagous bats, known as vampires, in Central and South America. Several years later, rabies was also detected in non-hematophagous bat species.13 Although RABV is found worldwide in several terrestrial hosts, its presence in bats is observed only in the Americas. In Europe, four different Lyssaviruses have been isolated from bats: European Bat Lyssavirus type 1 (EBLV-1) and European Bat Lyssavirus type 2 (EBLV-2), Bokeloh Bat Lyssavirus (BBLV) and West Caucasian Bat Virus (WCBV).14 Recently, a new putative Lyssavirus in bat, named Lleida Bat Lyssavirus (LLBV), was found in Spain.15 To date, no human exposure to LLBV has been reported. EBLV-1, with the sub-types EBLV-1a and EBLV-1b, is the most isolated type throughout Europe. In addition, spillover infections by EBLV-1 in other mammals were also observed.14 The type 2 of EBLV is believed to be less virulent than type 113 and is found less frequently being present only in few countries and human contamination has been reported only in two cases.14 Two other members of this family are found in bats but significantly less frequently than the previous ones: BBLV isolated in Germany and France17; WCBV isolated once in the Caucasus Mountains but also detected in Kenya in seropositive bats, suggesting a greater geographical distribution.18 Australian Bat Lyssavirus (ABLV) is the first endemic lyssavirus identified in Australia and is phylogenetically related to RABV and EBLV1.19 ABLV has been identified in all flying fox species on Australia’s land mass. Three fatal human infections by ABLV have been reported. Additionally, other viruses of this family detected in bats are summarized in The first report of a transmission of a viral disease from bats to humans was in 1911 and related to the rabies virus (RABV) belonging to the Paramyxoviridae constitute a wide viral family that includes human and animal pathogens. Several bat-borne paramyxoviruses have been recognized such as parainfluenza type 2 virus, Mapuera, Menangle and Tioman viruses and two infectious agents of emerging diseases, such as Nipah and Hendra viruses.20Nipah and Hendra viruses, classified as the genus Henipavirus, are capable of causing severe, potentially fatal diseases in humans.20 Fruit bats of the Pteropus genus are the common reservoir hosts of the Nipah and Hendra viruses.20Nipah virus (NiV) first emerged in 1998 in Malaysia, causing an outbreak of respiratory illness and encephalitis in pigs.21 Pig-to-human transmission of Nipah virus – associated with severe febrile encephalitis – was described and it was thought to occur through close contact with infected animals. Although uncommon, human-to-human transmission of virus was also described.21 In two other outbreaks in Bangladesh and India, an intermediate animal host was not identified, suggesting bat-to-human and human-to-human transmissions.Hendra virus (HeV) causes a fatal respiratory disease in both humans and horses.22 Several outbreaks of HeV have occurred in Australia. Horse is the intermediate host and the virus is likely transmitted via ingestion of feed, pasture or water contaminated with urine, saliva and feces of infected bats. Horse-to-human transmission occurs when there is close contact with ill animals.20 To date, human-to-human transmission has not been observed.Coronaviruses (CoVs) prior to the SARS outbreak were only known to be the second cause of the common cold after rhinoviruses. At least four different species can cause mild, self-limiting upper respiratory tract infections in humans: alphacoronaviruses HCoV-229E and HCoV-NL63, and betacoronaviruses HCoV-HKU1 and HCoV-OC43. More recently, two more additional pathogenic human-CoV were identified: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV).23 SARS-CoV was first identified in China in February 2003, and 4 months later, >8000 cases had been reported with about 800 deaths in 27 different countries worldwide.24 SARS-CoV has a wide host range and it is associated with wildlife meat industry. The natural history of the virus involves bats as primary hosts that then transmitted it to the intermediate amplifying hosts – as mask palm civets and raccoon dogs – that then could spread it to humans.25 Human-to-human transmission follows and can lead to large numbers of infected patients and is considered the main route of transmission in large-scale epidemics.927 Several CoVs have been identified in insectivorous and frugivorous bat species in various countries, indicating that bats may represent an important reservoir of these viruses.23 MERS-CoV was first identified in Saudi Arabia in 2012 and then spread to other countries causing hundreds of deaths.28 Clinical features of MERS-CoV are similar to SARS-CoV, although this virus has also been associated with several extrapulmonary manifestations, such as severe renal complications. Recent studies have indicated that dromedary camels may be the intermediate hosts and potential source of the virus for humans.29 In addition, the first experimental infection of bats with MERS-CoV has been described. The virus maintains the ability to replicate in the host without clinical signs of disease, supporting the general hypothesis that bats are the ancestral reservoir for MERS-CoV.30 Human-to-human transmission has also been reported. Based on epidemiological data, both animal-to-human and human-to-human transmission are considered to be important elements in MERS outbreak.26MERS-CoV is phylogenetically related to SARS-CoV and share with SARS-CoV the origin in bats.Ebolavirus and Marburgvirus are two genera of the family Filoviridae, responsible for severe, often fatal, hemorrhagic fever diseases in humans and other primates.31 The first report on the Marburgvirus first was in 196732 in German laboratory workers in Marburg who contacted it from African monkeys imported from Uganda. In 1976, a virus with similar characteristics but immunologically distinct was isolated in the Northern Democratic Republic of Congo and it was named Ebolavirus.32 Both viruses have generated several epidemics during the past years.31 Recently, in 2014, the largest ever registered Ebola epidemic started in West Africa and has affected several countries with >10 000 confirmed cases and thousands of deaths . The natural reservoirs for Marburgvirus and Ebolavirus are both fruit and insectivorous bat species, indicating that these filoviruses are multihost parasites.33 The virus is transmitted to humans through contact with body fluids – mainly blood and feces – and dead bodies of infected bats. Other animals such as monkeys and apes can also develop the disease and in turn transmit it to humans. Epidemics usually are a consequence of human-to-human transmission of the virus  and neuraminidase (N). Influenza A virus is an uncommon promiscuous virus with a wide host range, including humans, pigs and birds. Recently, two new subtypes evolutionarily distinct from all others – H17N10 and H18N11 – were detected in different fruit bat species in Central and South America.36 It has been observed that, although H17N10 subtype is phylogenetically separate from all other subtypes, the virus genome is compatible with genetic exchange with human influenza A viruses, suggesting a potential re-assortment capability between subtypes and the consequent ability to generate highly pathogenic hybrid forms.35 More recently, serological evidence of influenza A virus subtypes other than H17N10 and H18N11 were reported in African frugivorous bats.37 In particular, about 30% antibody detection rate was found against avian H9 subtype known to cause infections in humans worldwide.38 These data, albeit preliminary, suggest that bats might represent asymptomatic mammalian carriers of influenza A viruses.37 Thus, similar to other pathogens, bats may represent a considerable reservoir for these viruses.Hantavirus genus (from Hantan river in South Korea) is constituted of several emerging segmented RNA viruses that can cause human infections, including severe and lethal diseases such as hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome.40 Rodents have long been believed to be the primary reservoirs of hantaviruses; however, a wider range of mammalian hosts, including insectivorous bats, has been reported.40 The evolutionary history of this genus is characterized by relatively frequent cross-species transmission that is also considered a major force in its evolution. The first hantavirus isolated from bats was the Hantaan virus, the etiological agent of hemorrhagic fever with renal syndrome.41 Successively, hantaviruses were identified in other bat species, but to date, however, no bat-to-human transmission of hantaviruses has been observed.39Mammalian orthoreovirus of the genus Orthoreovirus can cause mild respiratory or gastrointestinal illness to severe diseases, including encephalitis and diarrhea. The virus is present in different serotypes throughout the world and has been isolated from several mammals, including humans.42Mammalian orthoreoviruses were also isolated in several bat species, suggesting an extensive distribution of the virus in these animals.44 Several evidence suggest that bats may act as the natural reservoir of these viruses.42 Although bat-origin orthoreoviruses have been isolated from human patients, the zoonotic potential of these viruses is still unclear.4546–48 An example are Poxviruses – important infectious agents of both humans and animals and capable of infecting multiple host species and to induce cross-species infections that were also recently identified in bats.49 Another example is the Dengue virus, an arthropod-borne virus belonging to the Flavivirus genus (Flaviviridae) that includes several relevant human pathogens associated with encephalitis and hemorrhagic fevers. Despite the fact that Flaviviridae are the second most frequent viruses found in bats and Dengue virus has been described in several bat species worldwide, the role of these animals in the dynamics of viral spreading remain insufficiently understood.50Several other mammalian viruses have been detected in bats for which the zoonotic potential or host range is unclear.51 Here we report some examples of bacteria responsible for common human and animal infections that have occasionally been detected in bats.Unlike viruses, bacteria in bats and their putative threat to humans remain poorly studied.52Bartonella sp. is a Gram-negative bacterium transmitted through the bite of hematophagous arthropod vectors. Several species have been identified in domestic and wild animals, including bats.53–55 Recently, two species of Bartonella – B. mayotimonensis and B. naantaliensis – were detected from both the peripheral blood of bats and in their ectoparasites, suggesting that bats might be a source of the human bacterial pathogens.54 More recently, it has been reported the presence of closely related Bartonella genotypes in fruit bats and their associated bat flies in Madagascar, suggesting the transmission of a potentially zoonotic pathogen by bat fly vectors.56Bartonellosis is a globally emerging zoonotic bacterial disease.Pasteurella is commonly spread among animals as part of the normal microbiota of the oral, nasopharyngeal and upper respiratory tract.57 This genus comprises opportunistic pathogen species that can cause endemic disease and are associated with epizootic outbreaks.57 Animal bites and nasal secretions are the most likely sources of transmission to humans. In bats, various Pasteurella species – mainly P. multocida – have been identified as the main pathogens of several localized and systemic infections.58 The predominant source of infections appears to be wounds caused by the bite of domestic predators. However, a recent study from Wisconsin in USA reported for the first time an outbreak of acute pasteurellosis from P. multocida in wild bats without associated traumatic injures.59Leptospira has worldwide distribution and its transmission to humans is primarily through exposure to water contaminated with the urine of infected animals.60 Bacterium harbors in several wild and domestic hosts, colonizes their kidneys and it is eliminated in their urine. The presence of Leptospira in bats has been demonstrated in several studies.62 However, the potential role of bats in human leptospirosis is questionable.64 In a case report, the patient’s history of bat exposure supports the idea that bats are a reservoir of the bacterium and can serve as a vector in disease transmission to humans.64Enterobacteriaceae family – responsible for a variety of human illnesses – were isolated from bats.65–67 A number of studies reported that Salmonella serotypes isolated from bats have similar characteristics to those found from livestock and humans, indicating that bats can be locally important in the epidemiology of salmonellosis in human and domestic livestock.66 Two of these serotypes, S. typhimurium and S. enteritidis, are a frequent cause of human and animal diseases.Several members of the Escherichia coli strain has also been frequently isolated from bats.66–68 It is to emphasize the high percentage of multiresistance of these class of pathogens to several classes of antimicrobials68 that is a major and increasing global health-care problem. Antimicrobial resistance was also observed in domestic and wild animals, with an increased incidence of resistance in both pathogenic and endogenous bacteria.69 Resistant pathogens can then be transmitted to humans and bats can therefore contribute to the spreading of resistant bacteria.Yersinia, Campylobacter, Vibrio – have been identified in bats, but their impact on these animals remains mostly unknown.51Several other genera – such as H. capsulatum is a dimorphic pathogenic fungus of mammals, which causes pulmonary and systemic infections in humans and it is acquired via inhalation of the fungal spores. This microorganism is commonly found in soil associated with great amounts of birds’ droppings or bats guano. Although bats are considered the main reservoir and dispersers of this fungus in the environment, their role in spreading H. capsulatum remains unclear.70 It has, however, been observed that subjects occupationally exposed to bat sites, such as miners, geologists or farmers who use bat guano as fertilizer, have high risk of infection and can develop severe clinical forms of histoplasmosis.71P. destructans that infects the skin of bats – especially the wings – during the winter months while they are in hibernation.72 Unlike other dermatophytes, which colonize the outer layer of the skin, P. destructans is able to invade the living tissue of the host causing the characteristic severe skin lesions.73 In addition P. destructans increases the frequency of periodic arousals in bats, resulting in premature consumption of stored fat essential to survive the winter leading to death within 4 months of infection.72 Recently, it has been observed that bacteria of the Pseudomonas genus – isolated from the skin of bats – inhibit the growth of the fungus in vitro.74 Additional in vivo studies will tell us whether in the future they could be used as biological control agents to protect bats exposed to P. destructans.Although implications in human health for this microorganism are not known, it is important to write a few words on an emerging fungal disease, named WNS, responsible for the deaths of millions of bats in North America. It is caused by the psychrophilic (cold-loving) fungus 75 Most of these are caused by zoonotic pathogens originating in wildlife and then spreading to humans. Bats are an important reservoir of several pathogenic agents, mainly viruses, and many of them have already caused disease outbreaks worldwide. The increasing rate of bat-associated infections is also supported by an expanding overlap between bat and human habitats. Recently, to increase the knowledge of bat-associated viruses, a database has been constructed (http://www.mgc.ac.cn/DBatVir).76 DbatVir analyzes the virome diversity of bats as well as the ecological and epidemiological data to examine and track current and future bat-related transmissible diseases. To date, DbatVir has collected information on 5717 bat-associated animal viruses detected from 207 bat species in 77 different countries (update on 2 march 2016). Strategies on surveillance and monitoring of disease outbreaks in bat populations need to be further developed, in particular where bats and humans are in close contact. Extensive studies are also needed to improve our understanding of bat–human interactions to design new control measures in future. Furthermore, the identification of new human pathogens requires a continuous study to monitor the potential impact of these animals in their diffusion.Emergence of new infectious diseases correlates with socio-economic, environmental and ecological factors and are a major public health problem as well as an important burden on economies worldwide.


Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures.The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales ( The major histocompatibility complex (MHC) is a multigene family that plays a fundamental role in the vertebrate immune response ,2. SpeciBalaenoptera physalus; sei whales, B. borealis; beluga, Delphinapterus leucas), which are characterized by lower levels of MHC variation than most terrestrial mammals surveyed to date [Lipotes vexillifer [Megaptera novaeangliae [Delphinapterus leucas eight DQB alleles in 233 individuals were reported [Balaenoptera physalus three alleles were found in 36 individuals [DQB locus has been reported in some marine species  by finding more than two alleles per individual [Presumably as a result of pathogen-mediated balancing selection, MHC genes are typically among the most polymorphic loci in vertebrate genomes ,2. Appar to date ,7–9. The to date ,9. Due txillifer  and Megaeangliae  have revividuals ; the freividuals . Thus, tividuals ,11 is lidividual ,11.DQB locus in a population of blue whales  from the Gulf of California, Mexico. This locus was chosen as among mysticetes the DQB locus is reportedly more variable than other commonly studied MHC genes such as the DRB gene, which seems to be more variable in odontocetes [DQB gene encodes the peptide binding region (PBR) of the DQ β chain [To explore determinants of variation at cetacean MHC loci in greater detail, we characterized patterns of variability and selection at the MHC Class II ntocetes ,7). Furt β chain ,10,11,13DQB locus for a subset of the individuals overwintering in the Gulf of California. Our analyses generate new insights into patterns of immunogenetic variability in these animals as well as the impacts of selection on cetacean MHC genes over multiple time scales.Although cosmopolitan in distribution, this species is divided into four geographically and genetically distinct subgroups: Northwest Atlantic, Indic Ocean, South Ocean, and East Pacific . After eBiopsy samples were collected under D. Gendron annual research permits issued by the Secretaría de Medio Ambiente Recursos Naturales y Pesca  from 1997 to 2001, and by the Dirección General de Vida Silvestre, Secretaría de Medio Ambiente y Recursos Naturales  from 2002 to 2012, which now represents the only approved Government institution to issue research permits on endangered species in Mexico. By issuing these annual research permits they approved the methods and the number of blue whale skin-blubber biopsies collection for each year of the permit. A more detailed description of biopsy sampling it could be found in Costa-Urrutia et al. 2013 [Balaenoptera musculus) over-wintering in the Gulf of California, Mexico, were genotyped at the MHC Class II DQB locus. Skin samples (5–10 mm) were obtained from Diane Gendron Laniel and colleagues at the Laboratorio de Ecología de Cetáceos y Quelonios, Centro Interdisciplinario de Ciencias Marinas (CICIMAR), Instituto Politécnico Nacional, Mexico. Samples were collected from 1999 to 2012 using a crossbow to shoot a small dart (distance ~ 10 m) at the whale, the tip of dart was designed to secure a skin-blubber biopsy from an individual before falling free so that the dart could be retrieved from the water, this procedure required no direct contact with the study animals [2 portion of each biopsy was isolated and stored in 70% EtOH at 4°C until sequencing analysis. The remainder of each biopsy was placed in 20% dimethylsulfoxide (DMSO), and then frozen in liquid nitrogen to create a tissue archive for the study population [Skin biopsies (35–40 mm) from 80 blue whales  following the protocol of the manufacturer.We examined a 172 bp fragment of exon 2 of the MHC Class II DQB locus was performed using primers DQB-F and DQB-R from Murray et al.[Amplification of the ay et al.. Per-samDQB exon 2 was limited to a single variant per sequencing reaction, all PCR products of the appropriate size (225 bp) were cloned using the TOPO® TA Cloning® kit (Invitrogen Inc.) following the instructions of the manufacturer. Sequencing of eight clones per PCR reaction is expected to have a high probability (p = 0.992) of detecting both alleles in heterozygous individuals [To insure that sequencing of the ividuals  and thusAll sequences were aligned and edited using the program Geneious .5.6.6 . To deteDQB locus, we performed BLAST searches [At the nucleotide level, we defined sequence variants as distinct alleles following the criteria of Kennedy et al. : variantsearches  using thDQB alleles identified for B. musculus followed Kennedy et al.[Bamu (corresponding to the two first letters of the genus and the species names) and the suffix GC, which represents the Gulf of California locality where the tissues samples were collected [DQB sequences generated during this study were accessioned into GenBank as numbers: KJ179618 to KJ179639.Nomenclature for the dy et al.; each alollected . DQB seqS) and non-synonymous (dN) base pair substitutions  for all codons as well as for the subset of codons corresponding to the PBR of this exon. Ratios greater than one (ω > 1) indicate an excess of non-synonymous substitutions relative to neutral expectations and such values are typically interpreted as evidence of positive selection [DQB locus [B. musculus. We calculated values of ω for the putative PBR following the above procedure. Significance tests of differences between ω values were conducted using the Z-test contained in MEGA 5.2 [Evidence of selection at the molecular level was assessed using three distinct procedures. First, we calculated rates of synonymous  tree reconstruction was conducted by using the matrix of distances compound as implemented in PAUP* 4.0b10 [DQB alleles from B. musculus and other cetaceans, we constructed a median-joining haplotype network using Network 4.6 [To assess evidence for trans-species conservation of  GenBank , DQB all* 4.0b10 , genetic* 4.0b10 . These a* 4.0b10 , the runwork 4.6 , which iwork 4.6 .DQB locus was identified from the 80 blue whales examined , were sequenced. More than two DQB alleles were found in 36 (45%) of the surveyed animals , suggesting that members of the population possessed multiple copies of this DQB locus.A total of 757 cloned PCR products  being the most similar[A BLAST search indicated that the allotypes detected in our population displayed 90% to 99% concordance with exon 2 of the t similar. VariatiDQB. At the amino acid level, our sequenced fragment contained 57 amino acids of DQB exon 2 as indicated by the P value (P> 3.05).We obtained a positive value of the Tajima’s D (D = 1.078), however statistics calculations did not show a significant departure from neutrality at p < 0.01) in the entire 172 bp DQB sequence. Otherwise comparisons of ω for the PBR and non-PBR portions of exon 2 revealed that 10 (71.4%) of the 14 amino acid changes detected occurred in the PBR. Within the PBR, ω was significantly greater than one , in contrast, ω did not differ significantly from neutral expectations  for the non-PBR portion of the exon  of this exon. The DQB locus in our study population appears to be subject to selection at the nucleotide level.Class II MHC loci are typically thought to be subject to strong positive selection due to their role in detecting and responding to pathogens–4. Our fDQB alleles from our study animals and other cetaceans revealed evidence of trans-specific preservation of alleles, particularly between blue and humpback whales. Similar trans-specific conservation of MHC alleles has been reported for a variety of mammalian species including non-human primates [DQB locus in blue whales from the Gulf of California have been subject to selection over multiple scales.Comparisons of primates , bank voprimates , and tucprimates  as well primates ,13,14,42L. vexillifer: 33 functional alleles in 18 individuals [T. truncates: 27 functional alleles in 48 individuals [B. musculus from the Gulf of California are characterized by considerable MHC variation, the population of fin whales  from this locality displays low levels of MHC variation [In contrast to our findings, studies of a number of species of cetaceans have reported limited variability at MHC loci ,7–9,11, ividuals ), and Atividuals . FurtherDQB locus per individual in our study population. None of the alleles detected contained insertions, deletions, or stop codons, suggesting that some of these copies might represent functional sequences. We consider that our findings suggest that one or more gene duplication events have resulted in multiple functional copies of DQB locus, this assumption supports the hypothesis that cetacean DQB sequences are paralogous genes separated by gene duplication events, which is thought to be one of the major forces for MHC evolution [DQB locus has been reported for several other cetacean species  as well indicus , Cervus  elaphus , Bubalus bubalis , Mastomytalensis ), thereftalensis ,41,49,50One factor that may contribute to interspecific differences in immunogenetic variability among marine mammals is demography. For example, the population of fin whales in the Gulf of California is resident in that area and is isolated from a population of conspecifics . The GulDQB locus in blue whales from the Gulf of California revealed levels of immunogenetic variability comparable to those of many terrestrial mammals. Further, our data revealed evidence of selection on this exon at both the nucleotide and trans-specific levels, thereby contradicting the hypothesis that marine taxa are subject to less intense selective pressures than terrestrial mammals. Instead, we suggest that differences in current and historical demography may better explain the differences in immunogenetic variability evident among marine mammals. Future studies of MHC variation in blue whales and other marine taxa will benefit from exploring differences in pathogen-induced selective pressures as a function of both habitat and variation in current and historical demography.Our analyses of variation at exon 2 of the


Cabbage leaf curl virus interacts with all three NIKs from Arabidopsis to suppress their kinase activity [Tomato golden mosaic virus), TCrLYV (Tomato crinkle leaf yellows virus) and ToYSV (Tomato yellow spot virus) [in vitro and in vivo, and interacts with the ribosomal proteins L10 (rpL10) and L18 (rpL18) [The NSP-interacting kinase (NIK) receptor-mediated antiviral signaling has been identified as a virulence target of the begomovirus nuclear shuttle protein (NSP) ,2. NSP sactivity . The NSPt virus) ,2. Severt virus) . Second,t virus) ,3. In adt virus) . NIK exh (rpL18) . NIK-med (rpL18) . The con (rpL18) ,7. Our dTo identify novel regulators of NIK-mediated defense response, we screened a two-hybrid library for partners of rpL10. We discovered a novel transcriptional factor, which interacts with rpL10 in the nucleus of plant cells to down-regulate the expression of ribosomal genes. These data are consistent with the observation that constitutive activation of the NIK receptor by replacing Thr-474 with aspartate impairs global translation in Arabidopsis and tomato transgenic lines and confers broad-spectrum tolerance to begomovirus infection. Our data also indicate that the NIK immune receptor-mediated antiviral signaling operates through a bipartite module. One defense signal-transducing branch is mediated by the regulated nucleocytoplasmic transport of ribosomal protein to impair translation and the second branch of the antiviral signaling transduces a typical defense signal through induction of the plant immune system. We will discuss novel insights into the regulation of the NIK-mediated antiviral signaling and an efficient-acquired defense against begomovirus by modulating the activity of the immune defense receptor NIK in tomato plants.


Cryptococcus neoformans and Cryptococcus gattii are responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Infections due to C. gattii have mainly been described in tropical and subtropical regions, but its adaptation to temperate regions was crucial in the species evolution and highlighted the importance of this pathogenic yeast in the context of disease. Cryptococcus gattii molecular type VGII has come to the forefront in connection with an on-going emergence in the Pacific North West of North America. Taking into account that previous work pointed towards South America as an origin of this species, the present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population in order to gain new insights into its origin and global dispersal from the South American continent using the ISHAM consensus MLST typing scheme. Our results corroborate the finding that the Brazilian C. gattii VGII population is highly diverse. The diversity is likely due to recombination generated from sexual reproduction, as evidenced by the presence of both mating types in clinical and environmental samples. The data presented herein strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil, Amazonia and the Northeast. Numerous genotypes represent a link between Brazil and other parts of the world reinforcing South America as the most likely origin of the C. gattii VGII subtypes and their subsequent global spread, including their dispersal into North America, where they caused a major emergence. Cryptococcus neoformans and Cryptococcus gattii are fungal agents responsible globally for almost one million cryptococcosis cases yearly, mostly in immunocompromised patients, such as those living with HIV. Cryptococcosis is a life-threatening mycosis, frequently causing meningoencephalitis. Infections due to C. gattii were originally described in tropical and subtropical regions, but its adaptation to temperate regions was highlighted by the emergence in the Pacific North West of North America by C. gattii molecular type VGII. The present work aimed to assess the genetic diversity within the Brazilian C. gattii VGII population to gain new insights into its origin and global dispersal from the South American continent using the ISHAM MLST consensus typing scheme. Our results corroborate that the Brazilian C. gattii VGII population is highly diverse, and strongly supports the emergence of highly virulent strains from ancestors in the Northern regions of Brazil. Numerous genotypes represent a link between Brazil and other parts of the world, and the isolates from the transitional ecological area in Northeast Brazil are the most likely ancestor lineages, translocating from caatinga/cerrado by adapting progressively throughout Amazonia in South America, and spread to the North American Pacific Northwest region and other parts of the world on multiple occasions. Cryptococcus neoformans and C. gattii, frequently spreading to the central nervous system causing meningoencephalitis, with a lethality rate of up to 70% within three months after diagnosis , 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Invitrogen), 2 mM magnesium cloride, 2.5 U Taq DNA polymerase (Invitrogen), and 50 ng of each primer. The amplification was carried out in a thermocycler  at 95°C for 3-min initial denaturation, 30 cycles at 94°C for 1 min, annealing at 57.5°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 7 min. The unique fragment corresponding to each mating type was visualized after 3% agarose gel electrophoresis at 100 V.The mating type was characterized by PCR of the pheromone genes using primers specific for MATalpha, MFalfaU (5’TTCACTGCCATCTTCACCACC 3’) in combination with MFalfaL (5’TCTAGGCGATGACACAAAGGG 3’); and for MATCTTT 3’) . AmplifiC. gattii VGII isolates, including 127 clinical and 18 environmental isolates, collected between 1989 and 2010 in 4 out of the 5 Brazilian regions: 1. Northeast (n = 39), including isolates from Piauí (PI) and Bahia (BA); 2. North (n = 38), including isolates from Pará (PA), Amazonas (AM) and Roraima (RR); 3. Southeast (n = 59), including isolates from Rio de Janeiro (RJ) and São Paulo (SP); and 4. Central-West (n = 9), including isolates from Mato Grosso do Sul (MS). No VGII isolates were collected in the South region  of the country. The individual isolate data are in A total of 145 CAP59 locus, 13 for GPD1, nine for LAC1, 11 for PLB1, 38 for SOD1, eight for URA5 and 34 for the IGS1 region. Based on the combined analysis of the seven loci, a total of 81 sequence types were observed  region (Hd = 0.981) with 31 STs, and the lowest one in the Central-West (CW) region (Hd = 0.889) with five STs , and the second one with 46 STs, including the VGIIb sub-genotype . No isolates of the third North American sub-genotype VGIIc (ST6) were identified in Brazil, but a closely related sequence type ST272 (strain 438BP) from MS, the CW region of Brazil was identified .Among the 81 sequence types identified in Brazil, 54 are represented by a single isolate. The most frequent subtype, ST40, accounts for 13 isolates found in the Central-West (CW) and Southeast (SE) regions, followed by ST20 (VGIIa) and ST5, which contained nine and seven isolates, respectively . In geneC. gattii isolates from this study, and another nine different Brazilian STs obtained from previously published data (C. gattii due to its link with the less virulent ST7 (VGIIb). In addition, three main groups are linked to CC 278: 1) ST301 and all its descendants, mainly present in the SE region of Brazil, which is a triple-locus variant (TLV)  of ST124; 2) ST277 and all its descendants, mainly present in the North (N) region of Brazil, which is a double-locus variant (DLV)  of ST7 (VGIIb); and 3) ST281 and all its descendants, a mixed group of strains from all regions of the country, which is a TLV  of ST278. The important role played by ST278 isolated from the semi-arid NE region was confirmed after addition of 34 STs from other countries  (ST325 and ST313), both isolated from clinical samples. Clonal complex 5 is represented by the ancestor ST5 and constituted of eight clinical and environmental isolates from the North of Brazil. The other three SLVs of ST5 are ST265, ST288, and ST296, all isolated from clinical and environmental samples. Clonal complex 20 is represented by five STs , being the ST20 (VGIIa) the founder ST of this complex and composed by isolates from the SE and N .K = 3 populations , which mainly derived from North America, Asia and Australia  were identified as mating type alpha, 10% (15/145) were mating type  alpha/a .SOD1 locus and four at the CAP59 locus (D’ < 0.2) (Random mating can be evidenced by the linkage disequilibrium (D), converging to zero. The SNPs present in the seven loci were used to detect the evidence of recombination separately. Pairwise Linkage Disequilibrium (D) between SNPs suggested at least six recombination events responsible for the polymorphism at the ’ < 0.2) . The oth’ < 0.2) .CAP59 and SOD1 (p<0.05)  in the Brazilian population.In order to confirm these results we applied two other recombination tests to our dataset. The strong reticulation in the networks and phi test implemented in the SplitsTree software for the single sequences also indicate recombination within the Brazilian VGII isolates for (p<0.05) . These rC. gattii in temperate North America in 1999, it has been recognized as a major agent of severe pulmonary and neurological infections in this region [reviewed in Since the unexpected emergence of cryptococcosis caused by the VGII subtype of C. gattii VGII isolates, presenting 81 MLST STs in 145 clinical and environmental isolates. In addition, a high level of haplotype diversity was observed, while also demonstrating a high degree of homoplasy, with the Consistency Index suggesting the absence of a selective genetic pressure. The patterns of the polymorphisms identified among the Brazilian strains surveyed in this study indicated a history of recombination for the genetic loci CAP59 and SOD1 . The persistence of successful STs, which are stable in space and time and most significant in cases of widespread adapted clones, may follow the features of clonal evolution which is defined as strongly restrained recombination [C. gattii isolates belonged to the sub-genotype VGIIb (11 out of 12) [Despite the high genetic diversity in the Brazilian bination . This habination , protozobination , and funbination . Linked bination  identifit of 12) , suggeste.g. outbreak strains), as the killing of the host, in the case of an opportunistic Cryptococcus infection, will not interfere with fungal cell propagation.Despite some well-adapted clonal isolates, the herein described population is recombining. The evolutionary processes, sex crossing and consequently recombination, generates new combinations of genes, some of which may increase adaptation of the population to harsh environments to increase the chance of their survival . On the e.g. as São Paulo and Rio de Janeiro (SE) are the biggest cities in the country, many people from other regions migrate to the these regions to find greater and better work possibilities. In order to check the influence of migration of C. gattii throughout the country, which could also be due to human migration, the clone corrected dataset was submitted to the admixture model in Structure and showed one basal population distributed all over the country , reinforcing the possibility of indoor infection, especially in wooden houses, very common in the northern part of Brazil, which was originally suggested by Brito-Santos et al. [The sequence type ST7 (VGIIb) has been found all over the world, including: Australia, Canada, China, Korea, Thailand and the USA , 51, 52.s et al. .C. gattii behaves as an endemic fungal pathogen that causes infection in apparently healthy individuals categorized as immunocompetent patients, and the predominant VGII genotype has been recognized for at least the last 20 years among clinical and environmental strains from those large regions [C. gattii population from South America, probably from the Amazon rainforest [In the North and Northeast of Brazil,  regions . The res regions  but is c regions , have moinforest , 16, 18.C. gattii VGII isolates in the current study strongly supports the possibility of the emergence of highly virulent strains in the N and NE regions of Brazil, associated with different biotopes, one with extremely humid forest in the North (the Amazon Forest) and the other with open and predominantly dry savanna formations in Northeast (brushwood known as “caatinga”). Between them, there is a transitional region, with overlapping areas of humid forest, less humid tropical savannah (known as “cerrado”) then the dry caatinga, best observed in the states of Piauí and Maranhão.The detection of high genetic diversity amongst Brazilian C. gattii VGII, which was previously placed in the Amazon region in the state Roraima (CFP 439/LMM645 from 1998), North of Brazil, by Hagen et al. [C. gattii life cycle under such variable climatic conditions. These findings show the ecological adaptability of VGII to spread to new habitats, allowing it to survive in dry and humid warm or cold climate.An important finding of the current study is the central role of the ST278, which is associated with a clinical isolate (CFP 243) from the state Piauí and other closely associated STs from the same area. It shifts the global origin of n et al. , 57 to tC. gattii populations, inducing recombination events (and/or hybridization). Human-induced land use and extensive trade of native wood from the Amazon rainforest are also possible drivers of geographical dispersal of propagules, and consequently, disease emergence events.The Brazilian Northeast and North are large geographical regions, which have been subject to extensive deforestation, leading to enormous landscape changes and the establishment of new settlements, disturbing original communities and related habitats, and causing a large-scale biodiversity loss. These habitat changes, shifts in species composition and other stress factors may affect the profile of Taking into account that the present study has, like all previous studies, a possible sampling bias, over-representing some STs while others are underrepresented, it is necessary to suggest further studies investigating other ecological niches, such as a variety of human inhabited places as well as environmental samples from other tropical countries.The results indicate that the isolates from the transitional ecological area in Northeast Brazil are the most likely ancestor lineages, translocating from caatinga/cerrado by adapting progressively throughout Amazonia in South America, and spread to the North American Pacific Northwest regions and other parts of the world on multiple occasions. This picture is intrinsically related to climatic changes and devastating human activities globally. Therefore, a multifocal origin for the outbreak lineages of cryptococcal infections must be considered.S1 Table(XLSX)Click here for additional data file.


Halorubrum strains highly susceptible to numerous different viruses (up to 26). This high virus sensitivity, the abundance of broad host range viruses, and the maintenance of infectivity over a period of one year suggest constant interplay of halophilic microorganisms and their viruses within an extreme environment.Hypersaline environments around the world are dominated by archaea and their viruses. To date, very little is known about these viruses and their interaction with the host strains when compared to bacterial and eukaryotic viruses. We performed the first culture-dependent temporal screening of haloarchaeal viruses and their hosts in the saltern of Samut Sakhon, Thailand, during two subsequent years . Altogether we obtained 36 haloarchaeal virus isolates and 36 archaeal strains, significantly increasing the number of known archaeal virus isolates. Interestingly, the morphological distribution of our temporal isolates  was similar to the outcome of our previous spatial survey supporting the observations of a global resemblance of halophilic microorganisms and their viruses. Myoviruses represented the most abundant virus morphotype with strikingly broad host ranges. The other viral morphotypes  were more host-specific. We also identified a group of  During the last couple of decades, archaeal viruses have become a fascinating field of research due to several unique discoveries considering virus morphology, genomics, life cycles, and virus-host interactions ,4,5,6,7.Euryarchaeota or Crenarchaeota phyla [Halobacteriaceae [9 virus-like_particles per milliliter have been reported for viruses in hypersaline waters [All the known archaeal viruses infect extremophiles belonging to either ta phyla . All theta phyla ,6. Virusta phyla ,5. The meriaceae ,9,10. Hae waters ,12,13. Ae waters ,15.Caudovirales) and eukaryotic herpes viruses, indicating their common ancestry [Comparison of viruses has had a jump forward when more virion structures are becoming available. The enormous number of viruses in the virosphere seems to fall into a rather small number of structure-based lineages. Viruses within a lineage may or may not have detectable sequence similarity but they share the common virion architecture and major capsid protein (MCP) fold. This would suggest that the number of individual virus morphotypes is limited due to restricted protein fold space ,19,20,21ancestry . The icoancestry ,19,20,21To date, in the advent of metagenomics and bioinformatics with high-throughput sequencing and data handling methods, culturing of microorganisms and their viruses is largely neglected. However, it is still essentially the only means to obtaining reliable structural (up to high resolution), functional, and molecular information about viruses. Giving the example of virus evolution, structural information extends much further than genomic information when searching similarity ,20 and rIsolation of viruses is also important for studying their specific interactions with the host organisms. In 2012, we performed the first large-scale spatial screening of haloviruses and their hosts introducing “the global network” of virus-host interactions spanning nine hypersaline environments located on different parts of the world . The obtIf hundreds of virus-host interactions can be detected between spatially distant extreme environments, how would this relate to virus-host dynamics within one environment during different years? To date, temporal screenings of halophilic microorganisms in hypersaline environments have only been performed by culture-independent analyses ,4,26,27.Halorubrum strains. However, other virus morphotypes, such as the pleomorphic viruses, are more specific to their hosts.In order to increase the number of known archaeal virus isolates and to study virus-host interactions in one hypersaline environment over time, we performed a temporal culture-dependent study of archaeal haloviruses and their hosts in the solar saltern of Samut Sakhon, Thailand, during two consecutive years, 2009 (Samut Sakhon II (SSII)) and 2010 (Samut Sakhon III (SSIII)). We isolated and purified 36 virus isolates, characterized their virions, and compared the virion morphotype distribution to the one obtained from the same environment during the spatial study (Samut Sakhon (SSI)) . FurtherThe samples were collected from a solar saltern in Samut Sakhon, Thailand, 13°32'N; 100°17'E, in November 2009 (sample SSII) and December 2010 (sample SSIII). Samples included salt water and crystals from saltern fields . The den2 × 6H2O, 35 g MgSO4 × 7H2O, 7 g KCl, 5 mL of 1 M CaCl2 × 2H2O and 80 mL of 1 M Tris-HCl (pH 7.2) per liter) was diluted to obtain 18, 20, or 23% SW in the top-agar layer, solid, and broth media, respectively. MGM also contained 5 g of peptone (Oxoid) and 1 g of Bacto yeast extract  per liter. Fourteen (for solid) and 4 g (for top-agar layer) of agar  or Bacto agar  were added per liter of media.All cells and viruses were grown aerobically at 37 °C in modified growth medium (MGM) ,32. The g, 3 min, 22 °C), and the supernatants (100 µL) were plated. The liquid samples were plated directly. All the plates were incubated for up to 21 days. The obtained colonies were colony-purified three consecutive times, and the whole cell protein patterns of pure cultures were analyzed by sodium dodecyl sulfate 16% polyacrylamide gel electrophoresis (SDS-PAGE) [All prokaryotic strains used in this study are presented in DS-PAGE) .http://www.geneious.com/) was used for sequence assembly, and assembled sequences were analyzed using BLASTN [For partial 16S rRNA gene sequencing, PCR was performed as described previously . The PCRg BLASTN  and clasg BLASTN . The seqg, 5 min, 22 °C), and 100 µL of the supernatant was mixed with 300 µL of cells at exponential or early stationary phase. Three ml of molten top agar (50–60 °C) were added and the mixture was plated on MGM-plates. After 2–5 days of incubation, plaques were picked and plaque-purified three consequent times. Virus stocks were prepared from confluent or semi-confluent plates. For virus purification, virus particles were precipitated from the stocks with polyethylene glycol 6000, subjected to rate-zonal sucrose gradient centrifugation and when appropriate to CsCl isopycnic density gradient centrifugation using 18% SW as a buffer, as described previously [All viruses used in this study are presented in Proteins of purified virions were analyzed by tricine-SDS-PAGE (14% acrylamide in the separation gel) . ProteinFor transmission electron microscopy, purified viruses were negatively stained with 1% (w/v) potassium phosphotungstate (pH 6.5) for 5 s or 3% (w/v) uranyl acetate (pH 4.5) for 30 s. The micrographs were taken with JEOL 1200 EX or JEOL 1400 electron microscopes operating at 80 kV .Halogranum sp. SS13-5 and Halorubrum sp. SS13-13) and culture collection strains against all virus isolates was determined by a spot-on-lawn test. Drops (10 µL) of undiluted and 1:100 diluted virus stocks were placed on the lawn that was prepared by mixing the early stationary growing strain (300 µL) and soft agar (3 mL). After 3–5 days of incubation plates were analyzed for the presence of growth inhibition. All positive results were verified by the plaque assay.Sensitivity of all isolated strains  during two consecutive years . The samples included salt crystals , and salt water (ρ = 1.02–1.15 g mllt field .−1), we first isolated halophilic prokaryotes from SSII and SSIII sample sets. The different strains were selected based on the colony appearance and whole cell protein pattern analysis by SDS-PAGE (data not shown). We classified all the unique isolates at the genus level, using a threshold of 95% 16S rRNA gene sequence identity [Using modified growth medium , Haloarcula (seven strains), Halogranum (six strains), Haloterrigena (four strains), Haloferax (three strains), Halobacterium (two strains), Halogeometricum (one strain), Halobellus (one strain), Natrinema (one strain), and Halolamina (one strain). Four genera, Halogranum, Halorubrum, Haloferax, and Haloarcula included isolates from both SSII and SSIII samples. All Halobellus and Halobacterium strains were found from SSII samples, while Haloterrigena and Natrinema strains originated from SSIII samples.The archaea included 19 and 17 isolates from SSII and SSIII samples, respectively. The majority of the strains (30 out of 36) were isolated from salt crystals and only six from salt water samples. On the basis of their partial 16S rRNA gene sequences, we constructed a maximum likelihood phylogenetic tree  that alsHalorubrum sp. SS1-3, SS5-4, and SP3-3 [Haloarcula hispanica, “Har. californiae”, Har.japonica, Har. marismortui, Har. quadrata, “Har. sinaiiensis”, Har. vallismortis, and Halorubrum sodomense.We used a culture-dependent approach in order to isolate viruses from SSII and SSIII samples on endogenous archaeal strains derived from the same samples (see above). To increase the likelihood of finding new viruses from SSII samples, we included three culture collection strains (nd SP3-3 ) that arHalorubrum or Haloarcula strains. Only six of the virus isolates were obtained from salt water samples. The rest were isolated from salt crystals suggesting that terrestrial hypersaline elements are rich in viruses. Most viruses formed plaques after three days of incubation, and the plaque morphologies were either clear , 36 out of 45 virus isolates were successfully purified. The remaining nine isolates could not be purified due to low number of infective particles or instability during purification , which has previously been described in detail [Virion morphotypes of the purified viruses (Nos. 1–36) were determined by TEM A–E. The n detail ,24. We an detail . Among aHalogranum sp. SS13-5 and Halorubrum sp. SS13-13 were not included, because these strains do not form an adequate lawn. No interactions between viruses and bacteria were observed, showing that all the tested viruses are archaea-specific.Using a spot-on-lawn test for preliminary screening and subsequent plaque assay to verify the positive results, we determined specific virus-host interactions. All together ~3000 virus-strain pairs were cross-tested: all isolated viruses 45;  against We detected 268 specific virus-host interactions among the 36 virus isolates with known morphotype . By grouHalorubrum strains. HCTV-12 (No. 21 from SSIII) had the broadest host range including 14 different strains from five genera  . Among siphoviruses, the highest EOP was always on the original host strain  with the same or one order of magnitude lower EOP  . The rest strain . Pleomorower EOP . The icower EOPs .Halolamina, Halogranum, Haloferax, Halogeometricum, or Natrinema genera. Although, originally all the viruses were isolated either on Halorubrum or Haloarcula strains, cross-testing (see above) revealed that also strains from the genera Halobacterium, Halobellus, and Haloterrigena were infected by the isolated myoviruses . Other strains had significantly less interactions with viruses: ~10% for Haloarcula , ~6% for Halobacterium , ~2% for Halobellus  and ~1.5% for Haloterrigena strains   .Halorubrum strains were infected by viruses and the average number of interactions per host strain was 15, while the corresponding numbers for Haloarcula, Halobacterium, Halobellus, and Haloterrigena strains were five, eight, five, and four, respectively. Apart from Halorubrum, Halobacterium, and Halobellus, the genera Haloarcula and Haloterrigena also included strains for which no interactions were detected  that were susceptible to numerous viruses (from 13 up to 26). Notably, these strains originated from samples SSII and SSIII or belonged to the culture collection. In addition, Halobacterium sp. SS6-4, Haloarcula sp. SS8-4, and “Haloarcula californiae” were sensitive to ten, eight, and 13 viruses, respectively. Only Halorubrum sp. SS7-4 was infected by viruses representing all four different morphotypes, whereas about half of the virus host strains were sensitive to viruses of only one morphotype , whereas SSIII strains had 47 interactions, and culture collection strains 81 interactions . ConsequHalorubrum strains (We revealed 30 and 39 endogenous virus-host interactions in SSII and SSIII samples, respectively, comprising ~25% of all interactions . The tot strains .Culture collection strains had numerous interactions with the virus isolates. There were 12 and 69 interactions with SSII and SSIII viruses, respectively, composing ~5% and ~26% of the total interactions . VirusesHalorubrum strains are taken into account, the number of interactions is 202. This comprises ~75% of all interactions, suggesting that, at least according to this culture-dependent study, myoviruses and Halorubrum strains are the most dominant entities in the virus-host interaction network of highly saline environments. No strong temporal correlations were observed in the virus-host interactions detected during the two sampling years (To summarize, most of the interactions were those of myoviruses. If only their interactions with ng years . It seemng years . A slighng years . The eleIn addition to the 268 specific virus-host interactions, 73 interactions were observed for the virus isolates with unknown morphotypes . These vTemporal isolation of haloviruses and their host organisms in one environment has not, to our knowledge, been performed in the past using a culture-dependent approach. In this survey, optimized production and purification protocols were developed for all the 36 obtained virus isolates. This study together with the spatial halovirus screening published in 2012 with its 45 isolated haloarchaeal viruses , almost Halorubrum isolates although this feature was also observed to vary among the closely related strains (Morphotype distribution of the viruses isolated here resembles the one obtained during the spatial survey , highlig strains .Several culture-independent studies have suggested that head-tailed viruses are scarce in hypersaline environments compared to, for example, the lemon-shaped virus-like particles ,12,52. KHalorubrum strains obtained from SSII samples, indicating that the presence of such strains might increase the number of obtained virus-host interactions. The phenomenon of many viruses attacking the culture collection strains, which originate from distant environments, supports the previous observations of global distribution of related microorganisms and their viruses in hypersaline environments [A closer glance at the temporal virus-host interaction network of culturable strains and viruses of the Samut Sakhon saltern  reveals ronments ,56,57,58Temporal fluctuations of viral populations studied by metagenomics in the hypersaline Lake Tyrell, Australia, represent the most extensive temporal culture-independent survey performed to date in hypersaline environments ,28. Halo


Temporary closure and disinfection can rapidly reduce levels of infectious virus in these settings. We assessed the effect of closing live poultry markets in China on influenza A(H7N9) virus detection and viability. Intensive sampling was carried out before, during, and after a 2-week citywide market closure; the markets were cleaned and disinfected at the beginning of the closure period. Swab samples were collected at different sites within the markets and tested for H7N9 by real-time reverse transcription PCR and culture. During the closure, H7N9 viral RNA detection and isolation rates in retail markets decreased by 79% (95% CI 64%–88%) and 92% (95% CI 58%–98%), respectively. However, viable H7N9 virus could be cultured from wastewater samples collected up to 2 days after the market closure began. Our findings indicates that poultry workers and the general population are constantly exposed to H7N9 virus at these markets and that market closure and disinfection rapidly reduces the amount of viable virus. Influenza A(H7N9) virus emerged in eastern China in March 2013; within 2 years, infections were confirmed in >550 persons and >200 persons had died . To limit potential market-specific variations in virus activity, enhanced surveillance sites were set up in Panyu district, which comprises one tenth the population of Guangzhou . SamplesDuring routine and enhanced surveillance, GZCDC collected environmental samples from poultry cages; the inner surface of defeathering machines; chopping boards; surfaces of processing tables; and barrels holding poultry meat, wastewater, and drinking water for chicken. Because each type of LPM has a different setup for poultry processing and sales, environmental samples were collected only from the relevant sampling sites available within the respective LPMs .Universal transport medium  was used to preserve the environmental samples, which were stored in a box with ice packs at 4°C and transported to the laboratory within 4 hours. A QIAamp Viral RNA Mini Kit  was used to extract viral RNA. Influenza A virus M gene and H7N9 virus hemagglutinin (HA) RNA were detected as described previously (http://www.megasoftware.net) by using a neighbor-joining method with 1,000 bootstrap replicates.The HA gene of isolated strains was amplified by rRT-PCR, and the products were sequenced by Life Technologies Inc.  as described previously .We tested the effects of market disinfection and closure on detection rates for H7N9 virus and for all AIVs at different times after these interventions were implemented. Logistic regression models were used, accounting for potential confounders such as specific markets and sampling sites. The effect of market disinfection and closure on H7N9 virus isolation rates was similarly tested, after accounting for missing data from H7N9 virus rRT-PCR–positive samples not available for culture, by using multiple imputation methods with 50 imputed datasets. Given the short study period, limited effects of meteorological variables on virus activity were assumed and not adjusted for in the model. We also compared virus detection rates between the different sampling sites. All statistical analyses were conducted by using R version 3.1.1 , by mutations at 3 sites . Phylogenetic analysis showed that the strains were genetically closer to the lineage in southern than in eastern China .We report RNA detection rates for H7N9 virus and for all AIVs as well as viable virus survival in retail and wholesale LPMs and a DPM in Guangzhou before, during, and after market closure and disinfection. Before interventions were implemented on February 15, 2014, detection rates were much higher in retail markets than wholesale markets; this finding is in keeping with the theory that AIVs amplify in retail LPMs  in water (We did not quantify the infectious virus load by titration, even when virus could be cultured at the first sampling time point after market closure; thus, it is possible that the virus titer had decreased compared with that during the preintervention period. It is not clear whether virus titers would have been sufficient to initiate reinfection of reintroduced naive poultry. Previous studies on H9N2 virus demonstrated substantial reduction in isolation rates after a ban on keeping poultry overnight at LPMs, suggesting that transmission can be interrupted by emptying the market overnight (We did not collect samples during days 3–5 days after implementation of the interventions. However, the estimated reduction in H7N9 virus and other AIV RNA detection rates by rRT-PCR within a day of the citywide market closure was ≈50% from enhanced and routine surveillance .We observed that all 3 types of poultry markets were recontaminated by H7N9 virus and other AIVs; immediately after markets were reopened, detection rates were as high as those before market closure. In contrast to the preintervention period, during the postintervention period, we found no difference in virus detection rates in the retail and wholesale markets. The reason for this is unclear. It is possible that the sudden closure of the LPMs led to a backlog of poultry in other temporary holding facilities, where mixing of poultry originating from different areas could have led to virus amplification before the poultry reentered the wholesale market. Aside from within-market interventions, tightening controls on poultry or shortening transportation time along the supply chain may be needed to further reduce virus load in LPMs. For the DPM, poultry may have been stored temporarily and prepared elsewhere to preserve freshness, which may have contributed to the unexpectedly high detection rates in this market.Market rest days, along with a series of other control strategies, have been shown to reduce circulation of low pathogenic AIV in the retail LPM setting (Although market closure has been demonstrated to be effective in reducing influenza infections in humans in China (Poultry workers in China still demonstrate relatively low awareness of the risk for H7N9 virus infection and compliance with measures to prevent virus transmission (Our study has some limitations. First, the unexpected timing of the poultry market closure shortened the preintervention baseline period and the enhanced surveillance period during which we could obtain samples from LPMs. Thus, the preintervention data may not fully reflect the normal situation, especially in wholesale markets . HoweverWe document the effect of market closures on survival of H7N9 virus in a natural LPM setting. Market closure and disinfection reduced H7N9 viral RNA contamination in the LPM environment by >70% and infectious virus by >90%. However, live virus could be detected for ≈2 days after the intervention, especially in wastewater sources, and H7N9 virus activity returned quickly to preintervention levels once markets reopened. The reason for this rebound requires further investigation to inform the design of more effective interventions. Given limited support from the general public for permanent closure of LPMs (