Cryptococcus gattii infections in southern California have been reported in patients with HIV/AIDS. In this study, we examined the molecular epidemiology, population structure, and virulence attributes of isolates collected from HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type, in contrast to the VGII molecular type isolates causing the North American Pacific Northwest outbreak. The global VGIII population structure can be divided into two molecular groups, VGIIIa and VGIIIb. Isolates from the Californian patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and produce abundant sexual spores that may serve as infectious propagules. The a and α VGIII MAT locus alleles are largely syntenic with limited rearrangements compared to the known VGI (a/α) and VGII (α) MAT loci, but each has unique characteristics including a distinct deletion flanking the 5′ VGIII MATa alleles and the α allele is more heterogeneous than the a allele. Our studies indicate that C. gattii VGIII is endemic in southern California, with other isolates originating from the neighboring regions of Mexico, and in rarer cases from Oregon and Washington state. Given that >1,000,000 cases of cryptococcal infection and >620,000 attributable mortalities occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases and highlight possible host differences among the C. gattii molecular types influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts. Cryptococcus, which infects >1,000,000 patients annually causing ∼1/3 of all AIDS-related deaths. Many infections are caused by C. neoformans, but the sibling species C. gattii infects both healthy individuals and HIV/AIDS patients and is causing an outbreak in the Pacific Northwest. Often, Cryptococcus is not identified at the species level, and C. gattii may therefore be more common than appreciated. We studied C. gattii isolates infecting HIV/AIDS patients in southern California and found they are unrelated to those causing the Pacific Northwest outbreak (molecular type VGIII rather than VGII). We show that within VGIII there are two subgroups, and one is more virulent than the other in animals. Our studies establish a foundation for research on C. gattii in HIV/AIDS patients. Given the global magnitude of infections, these findings have significant public health implications.Infections that were once uncommon have become considerable public health threats due to the increased number of patients with weakened immunity, including a large HIV infected population. Among the emerging infections in the HIV/AIDS population are those caused by fungi. Fungi are distinct from viral or bacterial pathogens and often cause skin (athletes foot), mouth (thrush), or vaginal (yeast) infections. Life-threatening infections of the lungs and brain are caused by  Cryptococcus species complex is comprised of two common fungal pathogens of humans and other animals: C. neoformans and C. gattiiC. neoformans is ubiquitously distributed worldwide and a common cause of meningitis in immunocompromised hosts C. gattii is more geographically restricted to tropical and subtropical regions, associated with eucalypts, Douglas fir, and other trees, and has a greater predilection for infecting immunocompetent hosts C. gattii can be subdivided into two serotypes (B and C) C. neoformansThe pathogenic C. neoformans, less is known about the epidemiology and ecology of C. gattii, especially molecular types VGIII and VGIV. VGIV is rare globally but has been reported to cause infections in sub-Saharan Africa AIDS patients Cryptococcus cases among HIV/AIDS patients, although sporadic C. gattii infections in HIV/AIDS patients have been reported from many global regions Compared to C. gattii in North America. However, C. gattii has been reported in southern California among an HIV/AIDS patient cohort and one AIDS patient in Mexico, and C. gattii VGIII/VGIV have been reported in clinical cases from Mexico C. gattii may be endemic in western North America.There are limited accounts for HIV/AIDS or VGIII/VGIV associated C. gattii isolate cohort are VGIII, and genotypic diversity is much greater than in the Pacific Northwest VGII outbreak. The genotypes cluster into two distinct groups . These findings suggest that VGIII may have been endemic in southern California for a longer period of time compared to the more clonal VGII Pacific Northwest population, or that VGIII is more actively recombining and/or mutating. The high levels of diversity, together with results suggesting recombination, support ongoing genetic exchange.Here we compared isolates from HIV/AIDS patients in southern California C. gattii molecular types a), unlike the exclusively α mating type Pacific Northwest outbreak a-α mating can promote recombination and yield infectious spores C. gattii VGIII MAT locus alleles. These findings expand on previous studies of the Cryptococcus MAT locus The VGIII lineage is highly fertile compared to other C. gattii is highly virulent in mice, the focus has been on genotypes causing disease in otherwise healthy hosts MAT locus plays in VGIII virulence. This was of interest, as previous studies have shown virulence differences in C. neoformans congenic isolates that differ only at MATWhile previous studies have shown C. gattii infections in southern California. Additionally, we show that these isolates are virulent in both murine and macrophage infection models Our studies reveal a complex population structure within the highly fertile VGIII molecular type, suggesting recombination in both the United States and globally via opposite and/or same-sex mating. We posit that genetic exchange and the formation of infectious spores may be contributing to an underlying endemic level of C. gattii isolates collected from the southern California C. gattii cohort a) (C. neoformans and C. gattii have been previously reported To characterize the molecular type of ohort a) , and no ohort a) , 2, and ohort a) , 2. AddiTo further examine the isolates, their MLST profiles were compared to an additional 32 VGIII isolates collected from multiple locations and sources . Based oWe next examined the distribution of the sequence types by constructing maximum likelihood (ML) dendrograms . This anC. gattii molecular types   . From thI, VGIV) . While tI, VGIV) .SXI2a was not informative as there is only one allele. For the other three loci , TCS analysis revealed that the CAP10 locus and TEF1 locus shared alleles appear ancestral, while the most parsimonious hypothesis is that the PLB1 shared allele was introgressed between VGIIIa and VGIIIb because another allele (PLB1-20) is assigned as the ancestral root  showed that the VGIIIa and VGIIIb subgroups are separated . These analyses were completed for the global population, and also individually for the VGIIIa and VGIIIb subgroups. Additionally, the dataset was analyzed using all 60 isolates, and then was analyzed as a clone-corrected set with only unique genotypes represented (n = 28). Clone correction is commonly used to examine fungal populations that are known to often undergo asexual reproduction and clonal blooms, with less frequent sexual reproduction and meiosis A produce p-values that if significant reject the null hypothesis of recombination. If the p-values are not significant, the null hypothesis cannot be rejected and recombination can be inferred. The analysis illustrates that although the null hypothesis is rejected in the analysis of all isolates, VGIIIa individual, VGIIIb individual, and the overall clone corrected dataset, the null hypothesis cannot be rejected in the analysis of the individual VGIIIa and VGIIIb subgroups when the dataset is clone corrected  shown in a isolates are fertile compared to α isolates  or C. neoformans var. grubii serotype A  was correlated with VGIIIa and VGIIIb molecular types with the exception of one VGIIIb strain harboring the SXIα allele 38, which contained the VGIIIa genotype at this region and could reflect recombination within MAT as a result of α-α mating  and fingerprints 2 and 7 of the α locus showed multiple genotypes that were not correlated with the subgroups.Fingerprinting analysis of the α allele . Distincα allele . Additioα mating . Other VE. camaldulensis tree in 1992 a isolates . These results indicated that pulmonary cryptococcosis and not CNS dissemination was the primary disease manifestation in this model and the likely cause of mice mortality observed (Although there were some differences in the dissemination pattern of CA1499 (high virulence) and CA1292 (moderate virulence) by histopathology, the quantification of yeasts from infected lungs at weekly intervals did not reveal any significant difference . This coobserved .Intracellular proliferation rates (IPRs) were determined for each strain examined in the murine model of infection. This assay is correlated positively with murine virulence, based on previous studies Pacific Northwest outbreak isolates C. neoformans serotype A C. gattii also has been shown to cause disease among AIDS patients in both sub-Saharan Africa and the US C. gattii serotype C VGIV molecular type C. gattii isolates were examined from a previously reported HIV/AIDS patient cohort in southern California C. gattii VGIII and VGIV isolates associated with HIV/AIDS patients is in stark contrast to numerous C. gattii infections among immunocompetent individuals caused by VGI/VGII isolates, and the ongoing VGII outbreak in the North American Pacific Northwest Globally the burden of cryptococcal disease is significant with approximately one million annual cases C. gattii strains were analyzed in the context of a global VGIII isolate collection to further characterize this molecular type. Analysis of 60 isolates at eight loci resolved two distinct subgroups designated VGIIIa and VGIIIb. In contrast to the Pacific Northwest VGII outbreak that is largely clonal with one genotype causing the majority of illness C. gattii among HIV/AIDS patients.The Californian HIV/AIDS patient cohort Phylogenetic analysis of the molecular types revealed that VGIIIa is basal to VGIIIb . Given tA. Our analyses support the hypothesis of recombination within the distinctive VGIII subgroups. This is consistent with the observed molecular differences between the two groups. Our population results, taken together with an increased number of highly fertile VGIIIb isolates in comparison to VGIIIa, suggest that the VGIIIb group may be more actively recombining. Furthermore, the two excluded hybrid isolates appear to be C. neoformans var. neoformans/C. gattii molecular type VGIIIb hybrids . The enhanced fertility ofn the VGIIIb subgroup may contribute to hybridization with C. neoformans.The ability for microbial pathogens to undergo sexual reproduction involving meiosis is unique to the eukaryotic lineage, including the parasites and fungi. Sex may play a significant role in several aspects of pathogenesis, including the generation of genetic diversity, the formation of invasive hyphae or spores, and direct links between the mating type locus and mating pathways with virulence Cryptococcus can complete a full sexual cycle in association with plants, leading to the production of infectious spores The ability to undergo sexual reproduction also has implications for the formation of infectious spores. Recent studies show that spores can initiate disseminated cryptococcal disease in the murine inhalation model of infection MAT alleles of C. gattii molecular types VGI and VGII are highly conserved, based on both DNA sequence similarity and gene synteny MATα locus alleles of VGI WM276, VGII R265, and VGIII NIH312 share full synteny . Based on findings in C. neoformans, recombination hotspots may flank the MAT locus alleles and thus have contributed to the flanking region deletions C. gattii MAT locus alleles showed a more dynamic MATa locus compared to the α locus. Given the significant rearrangement of the MATa locus, and the deletion or exclusion of genes from the locus and flanking region, it appears that the VGIII MATa population has experienced increased genetic variation. Deletion or exclusion of genes from the MAT locus and flanking regions may be an indication of expansion or contraction of the VGIII MATa locus. The finding of a SXI2a remnant conserved in C. gattii may signal a previous tetrapolar mating type state, consistent with the prevailing models and studies in related Cryptococcus species . However, an additional polymorphic marker within the MATα locus is needed to address the latter hypothesis.Within VGIII, fingerprinting analysis revealed a monomorphic C. neoformans, strains increase in virulence with an associated decrease in capsule size Following the definition of two well-supported lineages including isolates from both mating types, we examined virulence characteristics associated with these properties. We found significant differences between phylogenetic subgroups. In both whole animal murine in vivo intranasal instillation and proliferation assays in murine derived macrophages, the VGIIIa subgroup is more virulent than the VGIIIb subgroup. These findings are significant and serve as a foundation for future studies to determine the molecular basis for these observed phenotypes. Additionally, from a public health and epidemiological standpoint, it may be useful to determine if isolates are VGIIIa or VGIIIb. Clinical studies would have to be conducted in coordination to ascertain if the molecular subgroup is associated with altered clinical manifestations or outcomes. Based on the histological examinations, our findings indicate that host tissue responses in combination with yeast cell multiplication and capsule induction are associated with the outcome of pulmonary cryptococcosis, similar to previous studies documenting that upon serial in vivo passage of C. gattii VGIII molecular type, which has historically been less studied than both the VGI and VGII molecular types. Our findings support two distinct lineages that might each be recombining, with the VGIIIa lineage showing higher levels of virulence in the models examined. Of significance, many of the isolates examined were from a cohort of HIV/AIDS patients in southern California. The high level of VGIII observed is in stark contrast to the Pacific Northwest VGII outbreak of VGII, in which the vast majority of cases reported are not associated with HIV/AIDS infected patients C. gattii may occur in two general patient settings: VGI/VGII in otherwise healthy hosts (>50%) or those treated with steroids, vs. VGIII/VGIV predominantly in HIV/AIDS patients. Moreover, C. gattii infections may cause a substantial unrecognized health burden. To address these aspects, both retrospective and prospective studies should be conducted to: 1) survey global isolate collections from HIV/AIDS patients and 2) assign species and molecular types to newly collected isolates from HIV/AIDS patients with cryptococcal infections.Our study provides a comprehensive molecular and phenotypic overview of the C. gattii. Melanin production was assayed by growth and dark pigmentation on Staib's niger seed medium; urease activity was detected by growth and alkaline pH change on Christensen's agar. These tests established that isolates were Cryptococcus (C. neoformans or C. gattii). Additionally, isolates were assayed for resistance to canavanine and utilization of glycine on L-canavanine, glycine, 2-bromothymol blue (CGB) agar. Growth on CGB agar indicates that isolates are canavanine resistant, and able to use glycine as a sole carbon source, triggering a bromothymol blue color reaction indicative of C. gattii. All CGB positive isolates were then grown under rich culture conditions prior to genomic DNA extraction and storage at −80°C in 25% glycerol. For genomic DNA isolation, the MasterPure Yeast DNA purification kit from Epicentre Biotechnologies was used.All isolates from southern California and other global isolates were screened to confirm that they were C. gattiiEach VGIII isolate examined in the analysis was subjected to multilocus sequence typing (MLST) For each isolate, a sequence type was defined as a sequence exhibiting a unique sequence profile, based on concatenation of the MLST markers. Each sequence type was confirmed to be unique by BLAST analysis of the NCBI GenBank database Mating assays were conducted on V8 media (pH = 5). Isolates were incubated at room temperature in the dark for 2–4 weeks in dry conditions. Fertility was assessed by light microscopic examination for hyphae, fused clamp cells, basidia, and basidiospore formation at the periphery and surface of the co-incubated mating patch. All mating assays were conducted in duplicate. If there were no signs of fertility after the four-week period, isolate pairs were scored as having no fertility when paired together.2  for 15 min at the critical point. The agar pieces were mounted and sealed with silver paint to ensure good conductivity. The samples were sputter coated with 50 Å of Au/Pd . Samples were held under vacuum conditions until viewed with a Jeol JSM 5900LV scanning electron microscope at 15 kV.SEM analysis was completed using protocols similar to those previously published MAT locus alleles were NIH312 (MATα) and B4546 (MATa). Isolates were grown on YPD media at 30°C, and library construction was performed according to previous studies MAT locus of C. gattii were annotated based on homology to the existing annotation in C. gattii VGII (strain R265) and VGI (strains WM276 and E566), and C. neoformans (H99 and JEC21). Certain gene annotations were modified using the FGENESH program (http://linux1.softberry.com/berry.phtml). Comparison of the MAT locus alleles among VGI, VGII, and VGIII strains was performed using BLASTn. The BLASTn results were parsed using a PERL script and imputed into the ACT program to construct synteny diagrams of the MAT locus alleles among C. gattii strains. In order to evaluate size polymorphisms within the MAT locus, 14 MATα strains representing all SXI1α alleles and 8 MATa strains were amplified using primers complementary to conserved sequences. Primers used for fingerprinting are listed in http://tools.neb.com/NEBcutter2/index.php). Fingerprints showing variability were sequenced to determine the nature of any size polymorphisms. Sequences from both forward and reverse strands were assembled with complete double strand coverage and manually edited using Sequencher version 4.8 (Gene Codes Corporation).The strains used for the construction of the Bacterial Artificial Chromosome (BAC) and fosmid libraries and analysis of the C. gattii strains belonging to the VGIII genotype was tested in a murine model of pulmonary cryptococcosis C. gattii strains were grown in YPD broth for 16 hours and washed with sterile physiological saline. Cells were counted with a hemocytometer and suspended at a concentration of 3.3×106 cells/ml. Five male BALB/c mice  in each group were first anesthetized with xylazine-ketamine mixture and then 30 ul of infectious inocula was gently dripped into their nares. The injected animals were observed for any overt signs of illness, and all morbid animals were promptly sacrificed by CO2 inhalation to minimize pain and suffering.The pathogenic potential of http://faculty.vassar.edu/lowry/utest.html). Progression of disease was also determined by weighing infected animals once every alternate day at the start of the infection and then every day until they were moribund. One half portion of the lung and brain tissues from sacrificed animals was cultured on YPD and Staib's niger seed agar for the recovery of cells to determine that infections were cryptococcal in origin.Data from all infected animals were used to determine Kaplan-Meyer survival curves using SAS software . For this statistical analysis with genotype information, the non-parametric Mann-Whitney U-test was applied, with further details in the statistical analysis section (C. gattii strains from four virulence groups comprising high virulence strains (CA1499), moderate virulence strains (CA1292), low virulence strains (WM161), and avirulent strains (CA2339), were chosen for further intranasal infection per procedure described previously. A total of six mice were infected for each test strain and groups of three mice were sacrificed at one and two-weeks post infection. The lungs and brains were removed aseptically, one half of each organ was fixed in 10% buffered formalin and Bouin's fixative, processed into paraffin blocks, sectioned, and stained with hematoxylin and eosin (H & E) and Mayer's mucicarmine for histopathological examination. The other halves of the lungs and brains were weighed, homogenized, diluted in PBS, and plated on YPD agar. Colonies were counted after incubation of the plates at 30°C for 4 days, and the results were expressed as colony forming unit (CFU) per gram of infected tissue. Results from organ load experiment were analyzed by student t-test, with significance determined at P≤0.05.To characterize virulence properties, representative The animal studies conducted were in full compliance with all of the guidelines set forth by the Wadsworth Center Institutional Animal Care and Use Committee (IACUC) and in full compliance with the United States Animal Welfare Act (Public Law 98–198). The Wadsworth Center IACUC approved all of the vertebrate studies. The studies were conducted in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).2O was added into wells to lyse macrophage cells. After 30 minutes, the intracellular yeast were released and collected. Another 200 µl dH2O was added to each well to collect the remaining yeast cells. The intracellular yeast were then mixed with Trypan Blue at a 1∶1 ratio and the live yeast cells were counted. For the subsequent five time points , intracellular cryptococcal cells were collected and independently counted with a hemocytometer.A proliferation assay was previously developed to monitor the intracellular proliferation rate (IPR) of individual strains (for a 72-hour period) following phagocytosis For each strain tested, the time course was repeated at least three independent times, using different batches of macrophages. The IPR value was calculated by dividing the maximum intracellular yeast number by the initial intracellular yeast number at T = 0. We confirmed that Trypan Blue stains 100% of the cryptococcal cells in a heat-killed culture, but only approximately 5% of cells from a standard overnight culture. Compared to a conventional colony counting method, this method was shown to be more sensitive in detecting the clustered yeast population or yeast cells undergoing budding.http://faculty.vassar.edu/lowry/utest.html). Regression analysis was used to measure the correlation between LT50 and IPR values, and an F-value of p<0.05 was considered to be a significant correlation, with R2, p values, and Pearson correlations also calculated and represented in the analysis.Time to 50% lethality (LT50) and median IPR values were used to assess the statistical significance between the VGIII subgroups and these respective values. For this statistical analysis, the non-parametric directional Mann-Whitney U-test was applied and values of p<0.025 were considered as statistically significant  and supporting bootstrap values of the sequence data from global VGIII isolates, with the exclusion of MAT locus linked markers (SXI1α/SXI2a). VGI, VGII, and VGIV out-groups are included.(TIFF)Click here for additional data file.Figure S2Additional haplotype networks for MLST markers not shown in . Alleles for each respective locus are indicated numerically. Orange coloration represents VGIIIa and green VGIIIb. Circles represent alleles extant in the population, and the smaller black circles represent alleles that have not been recovered, or which may no longer be extant in the population. Each line connected to an object represents one postulated evolutionary event, with the squared allele representing the posited ancestral allele.(TIFF)Click here for additional data file.Figure S3Additional informative paired allele graphs from VGIII global isolates. An hourglass shape indicates the presence of all four possible pairs of alleles and serves as evidence of recombination.(PDF)Click here for additional data file.Figure S4Phenotypic analysis of VGIII MATa isolates related to the loss of the FCY1 gene from the MAT locus. Results indicate that FCY1 or another gene is still functioning to retain expected WT phenotypes involving sensitivity to the antifungal agent 5-Fluorocytosine (5-FC) and utilization of cytosine as a sole nitrogen source. Control strains include S. cerevisiae WT (S90) and an fcy1Δ mutant strain (S109) that is resistant to 5-FC and unable to utilize cytosine as a sole nitrogen source, and wild type control strains for C. neoformans var. grubii (H99) and C. neoformans var. neoformans (JEC21).(TIFF)Click here for additional data file.Figure S5Molecular analysis of VGIII MATa isolates related to the loss of the FCY1 and FAO1 genes from the MAT locus and the truncation of the UAP1 gene. A) List of strains. B) Southern blot analysis results indicate that the FCY1 gene may no longer be present in the genome, or that the gene may have undergone accelerated evolution and thus not be detected by hybridization to the probe that is based on the VGIIIα (NIH312) gene sequence. C) GPD1 control for Southern blot. D–F) PCR analysis for FCY1, FAO1, and UAP1, respectively, indicating a loss of the genes from the VGIII MATa genomes, or that the genes might have undergone accelerated evolution and thus not be detected with the oligonucleotide primers used.(TIFF)Click here for additional data file.Figure S6Remnant of SXI2a in VGIII α isolates. A) The grey shading indicates intergenic MAT sequence, while the black line indicates sequence outside of the MAT locus allele. The homology of the SXI2a remnant is indicated on the figure. B) A model for the evolution of the remnant based on partial gene loss in C. gattii and complete gene loss in C. neoformans. C) A model for the evolution of the remnant based on gene conversion within C. gattii.(TIFF)Click here for additional data file.Table S1Summary of mating assays and fertility. Top four isolates for fertility from each mating type indicate isolates that mated with the largest number of opposite mating type partners.(XLSX)Click here for additional data file.Table S2Details of fingerprinting regions with digestion information for MAT PCR products that were subject to restriction enzyme digestion.(XLSX)Click here for additional data file.Table S3Primers used in the study.(XLSX)Click here for additional data file.Table S4GenBank accession numbers for all of the MLST alleles represented in the text and figures, and sequences of the mating type loci.(XLSX)Click here for additional data file.Table S5Detailed sequence type information from  and (XLSX)Click here for additional data file.Table S6Strains used in this study.(XLSX)Click here for additional data file.


Herpesviruses are a major health concern for numerous organisms, including humans, causing both acute and chronic infections recurrent over an individual's lifespan. Marek's disease virus (MDV) is a highly contagious herpesvirus which causes a neoplastic condition in chicken populations. Several vertebrate-infecting herpesviruses have been shown to exist in an integrated state during latent periods of infection. However the status of MDV during latency has been a topic of debate.Here we employed high-resolution multi-color fluorescence in situ hybridization (FISH) to show integration of MDV at the telomeres of chicken chromosomes. Cytogenomic mapping of the chromosomal integrations allowed us to examine the clonal relationships among lymphomas within individuals, whereas analysis of tumors from multiple individuals indicated the potential for chromosomal preferences.Our data highlight that substantive genome-level interactions between the virus and host exist, and merit consideration for their potential impact and role in key aspects of herpesvirus pathobiology including infection, latency, cellular transformation, latency-breaks and viral evolution. Herpesviridae, genus Mardivirus, species Gallid herpesvirus 2) also exhibits oncogenic properties inducing lymphoid tumors in chickens.Herpesviruses are a large group of DNA viruses that cause disease in many organisms ,2. The aMDV rapidly infects B cells and subsequently transforms T cells during pathogenesis of the highly contagious neoplastic disease of chickens known as Marek's disease (MD). Since discovery , MD has A fascinating biological property that MDV shares with all herpesviruses is the ability to persist indefinitely in the host and evade immune response by establishing a latent infection. The molecular mechanisms controlling lytic replication and latent persistence of herpesviruses have been the subject of intense study in recent years, aided largely by advances made in the field of viral genetics. Although new information has emerged from these studies regarding mechanisms of the lytic cycle, factors controlling latency and tumorigenesis remain poorly understood. MDV presents a unique model system in which to explore the mechanisms of herpesvirus latency and virus-induced tumor formation.Another intriguing aspect of herpesvirus infection is that both episomal and linear-integrated forms of the virus are present in the host . One conTo resolve whether and where MDV integrates in the host chicken genome we employed high-resolution multi-color fluorescence in situ hybridization (FISH) using the tools developed from both the MDV and chicken sequencing efforts. We investigated the details of integration on both intra-and inter-chromosomal levels by mapping the location of MDV integrations. In depth analysis of MDV integration sites allowed us to investigate several specific objectives, including the clonal composition of MDV tumors and integration site preference of the virus. We show here that MDV specifically integrates into the telomeric regions of chicken chromosomes in MDV-induced clonal lymphomas.5 X 71,) . VirMapping lineage analysis indicates that four (of six) birds had multiple tumors of monoclonal origin, inclusive of one individual (Bird 1) showing a derived cell population Figure , which iThe indications for predominately, but not exclusively, monoclonal tumor origins, as well as derived cell populations within the MDV lymphomas, illustrates the complexity of tumor biology and the potentially diverse strategies employed by the herpesvirus. Understanding the timeframe during pathogenesis when MDV integrates into the chicken genome will be essential to assessing both the utility of integration profiles as a marker for clonality and the significance of integration on the latency cycle. In addition, further investigation of the effect of viral integration on cellular transformation is necessary. Integration may have a profound effect on transformation, or it may simply serve as a mechanism of maintaining the latent genome. We are currently investigating the stages at which MDV integrates, from early infection to late oncogenesis, and have preliminary indications that MDV integrates prior to transformation and tumorigenesis. Further research on the molecular mechanism for telomere-targeting of MDV integration should provide new insight regarding the role of telomeres in aspects of host genome stability and also viral evolution.The authors declare that they have no competing interests.CMR, HDH, HHC, and MED conceived of and designed the research project. CMR conducted the experiments. CMR and MED wrote the manuscript. All authors have read and approved the final manuscript.Hybridization of the MDV BAC probe to uninfected embryonic chicken cells. The MDV BAC was hybridized to chromosomes from uninfected chicken embryo fibroblasts. As shown, the MDV BAC probe did not hybridize to uninfected chicken cells, i.e., there are no FISH signals observed. Scale bar, 5 μmClick here for fileFISH comparison between two probes: the MDV-BAC and MDV cosmid clones. Panel A displays tumor 10-T1 labeled with the MDV cosmid clones (green) with a total of five integration sites indicated by arrowheads. Panel B displays tumor 10-T1 labeled with the full-genome MDV BAC probe (red) also with a total of five integration sites. Scale bar, 5 μmClick here for file


The significant biodiversity found in Brazil is a potential for the emergence of new zoonoses. Study in some places of the world suggest of the presence to hantavirus in tissues of bats. Researches of hantavirus in wildlife, out rodents, are very scarce in Brazil. Therefore we decided to investigate in tissues of different species of wild animals captured in the same region where rodents were detected positive for this virus. The present work analyzed ninety-one animals  from a region of the Atlantic forest in Biritiba Mirin City, São Paulo State, Brazil. Lungs and kidneys were used for RNA extraction.Micoureus paraguayanus, Monodelphis ihering, and Didelphis aurita) as well in two species of bats (Diphylla ecaudata and Anoura caudifer) is of significance because these new hosts could represent an important virus reservoirs.The samples were screened for evidence of hantavirus infection by SYBR-Green-based real-time RT-PCR. Sixteen samples positive were encountered among the wild rodents, bats, and opossums. The detection of hantavirus in the lungs and kidneys of three marsupial species (The analysis of nucleotide sequences of the partial S segment revealed that these genes were more related to the Araraquara virus strains. This work reinforces the importance of studying hantavirus in different animal species and performing a continued surveillance before this virus spreads in new hosts and generated serious problems in public health. The principal natural known reservoirs of the hantavirus are wild rodents. Studies illustrate that rodents infected with hantaviruses are stable reservoirs, shedding the virus in urine, saliva and faeces, but do not show overt signs of disease . The sigIn 2001, the region around Biritiba Mirin  experienced severe environmental degradation by the clearing of approximately 11.4 km sq of rain forest. This area has been flooded for dam construction and the wildlife is forced onto smaller tracts of land concentrating the potential disease reservoirs. Additionally, other forest areas are being illegally cleared and used for agricultural purposes increasing contact between wild animals and humans and likewise the concern of health workers.The present work analyzed ninety-one animals  from a region of the Atlantic rain forest in Biritiba Mirin City (23°34’28´´ S -46°02’18´´W), Sao Paulo State, Brazil. Bats were captured using mist nets strategically placed between 30 cm and 2 meters high arranged in parallel inside the forest during the night. The rodents and opossums were captured using Sherman and Tomahawk traps following the methodology previously described [Lung, kidney, spleen, liver, and heart tissues as well as urine samples were taken from each animal, following protocols approved by the Ethics Committee on Animal Experimentation of the Institute of Biomedical Sciences, University of São Paulo and with the permission of Brazilian Institute of Environment and Renewable Natural Resources . All other laboratory manipulations were performed inside the BSL 3 following standard operational procedures ,6.MegAlign program of DNASTAR package .RNA was extracted from the lungs and kidneys of all captured animals using RNA extraction kit , then amplified and detected by SYBR Green-based real-time RT-PCR  using specific primers previously described to amplify 141 bp fragment of the S segment . ResultsOligoryzomys nigripes, Oryzomys sp., Akodon sp., Tapthomys nigrita, Juliomys sp.; three species of opossum ; two hematophagous bats (Diphilla ecaudata) and one nectivorous bat (Anoura caudifer) , including: ten rodents belonging to five genera . The high similarity between the strains and the formation of a clade indicates that they are very close and share a common ancestor.Sequences obtained in the present study shared 93 to 95% nucleotide identity with the S segment of the Araraquara virus and Paranoa virus, forming a group nearest (Araraquara-like virus). Accession numbers (GenBank): Cases of human hantavirus infections continue to be reported in São Paulo State, and they represent an especially serious problem in newly occupied and deforested areas. The constant aggression against the natural environment, sudden climatic changes could alter the population densities of rodents and increase the rate of viral dissemination.In 2003, two months after we finished the sampling, local newspapers (Biritiba Mirim and region), reported that a farm worker died from hantavirus pulmonary syndrome (HPS) in the same region where the material was collected for the present study.O. nigripes specimen where as Araraquara- like virus were found in O. nigripes, A. montensis and others species [Necromys lasiurus, O. nigripes and Akodon sp. were considered potential natural reservoirs in Brazil [Previously work performed to validate this molecular surveillance of hantavirus in natural reservoirs revealed the presence of hantavirus Juquitiba-like in one  species . This ren Brazil .Hantaviruses are highly host-specific ; infectiRhinolophus ferrumequinum and Eptesicus serotinus[Neoromicia nanus), captured in West Africa, in June 2011 [The presence of hantavirus in tissues of bat was reported in Korea in 1993, with viral antigens being isolated from the lungs and kidneys of the bats serotinus,13. ReceMicoureus paraguayanus, Monodelphis ihering, and Didelphis aurita) as well in two species of bats (Diphylla ecaudata and Anoura caudifer) is of significance because these new hosts could represent an important virus reservoirs.The detection of hantavirus in the lungs and kidneys of three marsupial species (Transmission of hantavirus to different species could occur if virus replication was linked to the feeding behavior of these animals, or if contact due to overlapping habitat use increased a period after significantly. This region has experienced severe environmental degradation, the amount of space and food available to these rodent populations has been greatly reduced. Alterations in the equilibrium of rodent populations and in the dynamics of their interactions with humans can determine the occurrence of outbreaks. This situation has been intensified by wide-spread and severe changes in local ecosystems during the last few decades . To makeThe presence of opossum and bats in the same area where wild infected rodents circulate could be related to the dissemination of this virus by contact with the feces and urine of these rodents. Moreover, after the revolutionary breakthrough of isolating Hantaan strain virus, at least 14 viruses have been confirmed, and more than 40 species of small mammals have been identified as reservoirs or sources of hantavirus infection in the world .Our finds demonstrate the circulation of hantavirus in bats and opossum in Brazil and suggest an interspecies transmission that could be explained by a possible spillover of hantavirus among the wild animal populations of Biritiba Mirim, followed by shedding. This work reinforces the importance of studying hantavirus in different animal species and performing a continued surveillance before this virus spreads in new hosts and generated serious problems in public health.The authors declare that they have no competing interest.AJ and TLM carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. HDA, LDA, OT, DLM, ACC and FS participated in the sequence alignment, collect of samples, and laboratory work. DEL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The classical symptoms of porcine epidemic diarrhea (PED) are acute diarrhea and dehydration. The isolated porcine epidemic diarrhea virus (PEDV) CH/GDGZ/2012 strain was obtained from the feces of diseased pigs in 2012 in southern China. We report the complete genome sequence of strain CH/GDGZ/2012, which might be useful for better understanding the molecular characteristics of this virus. Alphacoronavirus, the family Coronaviridae, and the order Nidovirales (Porcine epidemic diarrhea virus (PEDV), which is an enveloped, positive-sense, single-stranded RNA virus, is classified as a member of the genus ovirales , 2.The whole genome of the PEDV strain CH/GDGZ/2012 was amplified by 21 sets of primers  and clonThe complete genome sequence of CH/GDGZ/2012 exhibits 96.6%, 97.4%, 97.9%, and 97.0% nucleotide homologies with the genomes of PEDV strains CV777, DR13, CH/FJND-3/2011, and CH/S, respectively . SimilarThese data may be useful for analyses of the epidemiology and evolutionary characteristics of PEDVs in southern China.KF384500.The complete genome sequence of PEDV CH/GDGZ/2012 strain has been deposited in GenBank under the accession no.


RFI) testing has increased selection pressure on biological efficiency in cattle. The objective of this study was to assess the association of the rumen microbiome in inefficient, positive RFI (p-RFI) and efficient, negative RFI (n-RFI) Brahman bulls grazing ‘Coastal’ bermudagrass [Cynodondactylon (L.) Pers.]under two levels of forage allowance (high and low stocking intensity). Sixteen Brahman bulls were previously fed in confinement for 70 d to determine the RFI phenotype. Bulls were then allotted 60 d stocking on bermudagrass pastures to estimate RFI using the n-alkane technique. At the conclusion of the grazing period, rumen liquid samples were collected from each bull by stomach tube to evaluate the rumen microbiome. Extraction of DNA, amplification of the V4-V6 region of the 16S rRNA gene, and 454 pyrosequencing were performed on each sample. After denoising the sequences, chimera checking, and quality trimming, 4,573 ± 1,287 sequences were generated per sample. Sequences were then assigned taxonomy from the Greengenes database using the RDP classifier. Overall, 67.5 and 22.9% of sequences were classified as Bacteroidetes and Firmicutes, respectively. Within the phylum Bacteroidetes, Prevotella was the most predominant genus and was observed in greater relative abundance in p-RFI bulls compared with n-RFI bulls (P = 0.01). In contrast, an unidentified Bacteroidales family was greater in relative abundance for n-RFI bulls than p-RFI . Ruminococcaceae was the third most abundant family in our samples, but it was not affected by RFI phenotype. No effect of stocking intensity was observed for bacterial taxa, but there was a tendency for alpha diversity and operational taxonomic unit richness to increase with lower stocking intensity. Results suggested the rumen microbiome of p-RFI Brahman bulls has greater levels of Prevotella, but the bacterial community composition was unaffected by stocking intensity.Residual feed intake ( RFI) RFIc); however, there is limited research related to RFI for grazing cattle (RFIg)where it may potentially be more useful to the industry Increasing feed and land costs are the greatest variable costs in beef cattle production n-RFI) have lower DMI than expected and may be classified as efficient; whereas less efficient, positive RFI animals (p-RFI) have greater feed intake at the same level of production. Digestibility and fermentation account for 19% of the variation in RFI The RFI has been correlated to lower heat production and methane emissions, and greater diet digestibility Bos indicus cattle compared with Bos TaurusThe use of DNA-based, high-throughput sequencing technologies provides a more encompassing view of the rumen microbiome though few efforts have been made to associate the rumen microbiome with RFI phenotypes. Large bacterial community changes in the rumen bacteria were not observed in dairy cows from divergent RFI phenotypes Cynodondactylon(L.) Pers.]pasturesat high or low stocking intensity  for 60 d. Stocking intensity (SI) was quantified and expressed by forage allowance (FA). Forage allowance was calculated by dividing total forage DM by the collective total animal BW per unit area of land. A single FA was calculated for each stocking rate per year.This “relationship between animal live weight and forage mass per unit area of the specific unit of land being grazed at any one time” is referred to as grazing pressure The experimental protocol was approved by the Institutional Animal Care and Use Committee at Texas A&M University (AUP 2010–053). This study was conducted at the Texas A&MAgriLife Research and Extension Center in Overton, Texas, in the Pineywoods vegetation zone in east Texas  during the summer of 2010. Purebred Brahman bulls (n = 33) were measured for ADG and DMI in confinement. The bulls were fed a commercially available growing ration in confinement using Calan gates at 2.8% BW. This % BW level was assessed to be 98–100% of the daily intake but with limited to no orts. The most efficient  and inefficient  bulls were separated into four groups with two p-RFI and two n-RFI bulls per group. They were randomly allotted into two replicate Coastal bermudagrass .Relative abundances of bacteria present >1% at phylum, family, or genus taxonomic level were logit transformed [z = log(p/(1-p))] to normally distribute residuals, where P = 0.06). An additional richness metric, ACE, also indicated a tendency for OTU richness to increase (P = 0.10) 122% for bulls grazing LSTK pastures compared with HSTK. The tendency to increase in observed OTUs with LSTK suggested the increase in alpha diversity (Shannon index) was due to greater OTU richness. No difference was observed in OTU richness or Shannon index between n-RFI and p-RFI bulls (P≥0.14). Comparison of microbiome communities by principal coordinate analysis did not reveal patterns related to RFIc phenotype or stocking intensity (data not shown).After pyrosequencing of 16 samples, 73,178 high-quality reads were used for downstream analysis and clustering of sequences yielded 4,906 OTUs at 97% similarity. Over 99% of OTU sequences aligned to phyla in the Greengenes database, while 72 and 43% of sequences were assigned at the family and genus taxonomic level, respectively.After rarefaction to 1,800 sequences per sample, greater OTUs were observed for bulls grazing LSTK bermudagrass pastures  to 78.4%  to 29.7%  in relative abundance. There was a tendency for an RFIc×SI interaction for the phylum Lentisphaerae(P = 0.09). No effects of SI or RFIc were observed at the phylum taxonomic level (P≥0.1).Across all samples, ectively . The avePrevotellaceae was the most prevalent identified family observed in all treatments representing more than 19% of all sequences. Greater relative abundance of Prevotellaceae was observed in p-RFI bulls (P = 0.01): a 169% increase compared with n-RFI bulls (33.2 vs. 19.6%). However, n-RFI bulls had an unidentified Bacteroidales family (1) in the greatest relative abundance overall and was 140% greater than p-RFI bulls . Family Ruminococcaceae was the third most prevalent family except for the p-RFI bulls on HSTK pastures. Ruminococcaceae relative abundance was not affected by SI or RFIc phenotype. Families BS11, Lachnospiraceae, Bacteroidaceae, Spirochaetaceae, as well as four unidentified families were not affected by SI or RFIc phenotype (P≥0.20). An RFIc×SI interaction was observed for families Paraprevotellaceae (P = 0.04) and Victivillaceae (P = 0.09); both families were observed in greater relative abundance in the HSTK treatment for the n-RFI phenotype bulls and the LSTK treatment for p-RFI bulls. At the genus taxonomic level, all sequences within family Prevotellaceaeclassified as Prevotella and were consistent with previously discussed effects (data not shown).Bacteroidetes  and stocking rate  had a significant impact on ADG Prevotellaceae (23.7% vs. 20.8) compared with n-RFI cows Fibrobacteraceaeand Entodinium weregreater in p-RFI cows, while Lachnospiraceaeand Dasytrichawere greater in n-RFI cows.However, principal coordinate analysis indicated rumen microbial communities were very similar overall. Beef heifers were evaluated for RFI in confinement and on pasture while qPCR was utilized to monitor changes in the bacterial community; Prevotella represented a greater proportion in p-RFI heifers and was the only bacteria related to RFI phenotype Similar to our findings, p-RFI dairy cows consuming fresh ryegrass  had a tendency for a greater relative abundance of Prevotella matching the overall average (26%) we observed Prevotellaceae was the predominant family (ranging from 19–23% in relative abundance), while 7–8% sequences were characterized by an unclassified Bacteroidales family and RuminococcaceaeCattle consuming bermudagrass hay (6% CP) hadsimilar proportions of Prevotella has been the dominant observed bacterial genus in the rumen microbiome Prevotella species include P. ruminicola, P. bryantii, P. albensis, and P. brevisPrevotella may be the most abundant genus observed, cultured species only accounted for a small fraction of the total Prevotella population Prevotella strains display highly varied genetic divergence Prevotella also have diverse functions. Prevotella species have a documented role in metabolism of starch, hemicellulose, pectin, and peptide or protein catabolism P. ruminicola and P. bryantii strain were sequenced to evaluate their genetic similarity Prevotellapylotypes Prevotella OTUs may be associated with efficient or inefficient cattle and may have different functional roles within the rumen microbiome.In many studies, Ruminococcaceaewere identified in the genus Ruminococcus. Two culturable species, R. flavefaciens and R. callidus, have been associated with cellulose and hemicellulose hydrolysis Ruminococcaceae unidentified at the genus level in this study may be genetically different from cultured ruminococci and possess non-cellulolytic functions.Although Ruminococci have been classically-studied in many culture-based experiments Prevotella was the genus observed in the greatest relative abundance and was greater in the rumen microbiome of p-RFI bulls. High forage allowance and resultant low stocking intensity tended to increase richness and alpha diversity within the rumen microbiome. Our results suggested rumen bacteria communities were largely similar between n-RFI and p-RFI Brahman bulls on HSTK and LSTK, though our sampling size and sequencing depth may have been a limiting factor to observing differences. Future isolation efforts and whole-genome sequencing of uncultured bacteria will provide greater understanding of their metabolic capabilities. Further defining the relationship between the rumen microbiome and efficient animals will allow for additional gains in livestock performance and productivity.In conclusion, Table S1Core microbiome OTUs detected in all n-RFI bulls.(DOCX)Click here for additional data file.Table S2Core microbiome OTUs detected in all p-RFI bulls.(DOCX)Click here for additional data file.


Influenza A virus has been detected in the blood of some infected individuals, and may pose a safety concern for collection, handling and transport of specimens for epidemiological and public health investigations if infectious virus is present in samples. Furthermore the effect of storage on virus stability and infectivity has not been well studied.50 assay when virus was stored in PBS, plasma, or buffy coats isolated from blood at 4°C.We examined the stability of novel pandemic influenza A (H1N1) virus RNA when the virus was stored in phosphate buffered saline (PBS), plasma, or buffy coated blood at either room temperature or 4°C using a sensitive Taqman RT-PCR assay. We also investigated virus infectivity using the EIDViral RNA stability was affected by the matrix used for storage. The recovery of viral RNA was highest when virus was stored in PBS with lower amounts being recovered from plasma and buffy coats at either room temperature or 4°C. Incubation time did not appear to be a major factor for viral RNA stability, although there was gradual decline after longer periods post-incubation. Both sample matrix and incubation time affected virus infectivity. The decay in virus infectivity was greatest in PBS followed by buffy coats and plasma. Virus infectivity was abolished in buffy coats at day 20 post-incubation when virus concentrations were low.These data indicate that encapsidated viral RNA was stable overall in all three liquid matrices at room temperature or 4°C although it was most stable in PBS; virus infectivity in buffy coats at 4°C decayed in a time dependent manner while it remained unchanged in plasma. These findings have implications for storage, handling and transport of blood derived samples from influenza patients for epidemiological and laboratory investigations. It should be noted that there is little known about influenza viremia, and whether influenza viruses can be transmitted by blood or blood derived samples. Influenza is a major cause of morbidity and mortality in the United States and worldwide. The possibility of an influenza pandemic has focused attention on the epidemiology and pathophysiology of influenza, including its potential for viremia in the acute phase of infection .Orthomyxoviridae family of RNA viruses. They contain eight segments of negative sense RNA  (FAM) at the 5' end, a non-fluorescent quencher and a minor groove binder (MGB), that served as a Tm enhancer, at the 3' end. The nucleic acids were amplified and detected in an automated TaqMan 7500 Analyzer by using the QuantiTect™ Probe RT-PCR kit . The 25-μl PCR mixture consisted of 100 nM each of primers and 100 nM probe. Following three thermal steps at 55°C for 5 min, 50°C for 30 min and 95°C for 10 min, 45 cycles of two-step PCR at 95°C for 15 s and at 60°C for 1 min were performed. The limit of detection was 1 fg of virus RNA per reaction with the TaqMan assay since the initial sample dilution was 1:10  and p < 0.01 (**) very significant.The unpaired Student's 6 to 3.55 × 104 EID50 of H1N1 virus in 10 μl of PBS were mixed with 130 μl of PBS, plasma, or buffy coat separately and held for different periods of time at room temperature. Nucleic acid was isolated and quantitated using the TaqMan RT-PCR assay. As shown in Figure Ten fold dilutions of H1N1 virus in 10 μl of PBS starting with 3.55 × 10Ten fold dilutions of H1N1 virus as described above were added to 130 μl of buffy coats and stored at room temperature, or 4 C for up to 48 h to test whether the viral RNA is degraded in buffy coats at different incubation temperatures. As shown in Figure To study the recovery of viral RNA stored in buffy coats at 4°C for different periods of time, H1N1 virus at the concentrations noted above was mixed with 130 μl of buffy coats, incubated 4°C for different periods of time. After isolation of nucleic acid and viral RNA detection with TaqMan RT-PCR assay, viral RNA copy number was not significantly changed during the incubation period at 4°C for all concentrations tested Figure .6, or 3.55 × 105 EID50 of H1N1 virus at 4°C, and found a similar trend in that there was no significant change of RNA recovery within each group of PBS, plasma or buffy coats  using the EID50 assay , virus infectivity was significantly abolished in PBS at day 10 postincubation relative to plasma and buffy coats  in PBS, or in buffy coats was reduced to zero at day 20 postincubation, the loss of virus infectivity in buffy coats was slower than in PBS at 10 day postincubation; and virus infectivity declined only gradually when stored in plasma, but was not abolished even at 40 day post-incubation  virus in PBS, plasma and buffy coats subjected to conditions often encountered in specimen handling, transport, and storage. We found that the RNA copy number did not change significantly when stored in PBS, buffy coats or plasma at room temperature up to 72 h or 4°C for up to 40 days. Although the recovery of viral RNA copy number was greater in PBS followed by plasma and buffy coats at either room temperature or 4°C, loss of virus infectivity was higher in PBS compared with buffy coats; infectivity in plasma did not change significantly under these conditions over time, especially at high doses of virus. We observed good correlation between H1N1 viral load and infectivity loss in PBS, plasma or buffy coats using the EIDThe authors declare that they have no competing interests.XW participated in the design, data collection and analysis, and drafted the manuscript.OZ and JZ participated in the data collection. ZY participated in the analysis and commented on drafts of the manuscript. IH conceived of the project, participated in the design and drafted the manuscript. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/354/prepubSupplement. Amplification curve for A (H1N1) influenza virus real-time RT-PCR assay. Shown are the serial dilutions of A/California/04/2009 (H1N1) RNA  with the FAM (novel swine-origin) probe. Reactions were run in duplicate. The forward primer is 5'-CGTCAGGCCCCCTCAAA-3', the reverse primer is 5'- TTTCCTGCAAAGACACTTTCCA-3', and the probe is 5'- CGAGATCGCGCAGAGA-3'.Click here for file


Resistance to oseltamivir was observed in influenza A pandemic (H1N1) 2009 virus isolated from an untreated person in Hong Kong, China. Investigations showed a resistant virus with the neuraminidase (NA) 274Y genotype in quasi-species from a nasopharyngeal aspirate. Monitoring for the naturally occurring NA 274Y mutation in this virus is necessary. Although oseltamivir has been widely used in persons infected with pandemic (H1N1) 2009 virus, resistance was not observed until recently. Three unrelated cases of resistance to oseltamivir were observed in Denmark, Japan, and Hong Kong  was detected in Norway in 2007. This virus has evolved into the dominant influenza A virus (H1N1) in humans  was positive for influenza A virus (H1N1) nucleoprotein by immunofluorescence. NPA specimens on days 1 and 5 were positive for influenza A virus (H1N1) M gene and swine-specific specific H1 gene by reverse transcription–PCR (RT-PCR). Samples obtained on days 6–8 were negative. Serum and midstream urine specimens obtained on day 2 were negative for influenza A virus (H1N1) M gene by RT-PCR.The patient refused antiviral therapy with oseltamivir because of fear of its potential side effects. She was then offered symptomatic treatment. Her clinical condition gradually improved and she was discharged on day 8 uneventfully.Influenza A pandemic (H1N1) 2009 virus was cultured from NPA. Subsequent drug susceptibility testing showed that this isolate was resistant to oseltamivir (50% inhibitory concentration 197.5 nM), but susceptible to zanamivir, as determined by enzymatic assay .To confirm whether the virus contained mutations associated with resistance to NA inhibitors, NA sequences from the day 1 NPA specimen and an MDCK cell isolate were examined. Viral RNA was extracted from NPA and MDCK cell supernatants by using reported procedures  residue (GenBank accession no. GQ351316). No other NA mutations known to be associated with oseltamivir resistance were observed. Further examination of sequences showed mixed populations (T/C) in the NA gene from the NPA specimen , panel AEstimation of 274H and 274Y populations in the NPA specimen was performed by cloning and sequencing PCR products. The NPA specimen contained approximately equal proportions of 274Y and 274H . Examination of sequences from the MDCK cell isolate showed predominantly the 274Y type, although a minor 274H peak was also observed , panel BResistance to NA inhibitors among seasonal strains of human influenza viruses  has been rare until recently. Development of resistance after oseltamivir treatment has occurred in 0.33%–5.5% of treated patients  was detected in Norway  2009 virus in the human population, combined with the inherent adamantane resistance of the virus, indicates that NA inhibitors constitute the primary treatment regimen for susceptible patient groups and those in whom severe diseases develop during the current pandemic. There is great concern that an oseltamivir-resistant variant of pandemic (H1N1) 2009 virus may emerge and circulate in a manner similar to oseltamivir-resistant seasonal influenza A virus (H1N1).The patient in this study was not treated with oseltamivir. Therefore it is unlikely that the 274Y mutation was drug-induced. Detection of mixed populations of 274Y and 274H in the NPA specimen before antiviral treatment suggests that the mutation occurs naturally, either before or during infection. Although no experimental data exist that show the growth properties of this resistant variant, examination of the quasi-species population in the cell culture–propagated virus isolate showed that the 274Y variant has become the dominant population. This finding implies that the 274Y mutation does not compromise replication of pandemic (H1N1) 2009 virus in vitro.Quarantine procedures adopted by the Hong Kong Special Administrative Region in China during the early containment phase might have limited transmission of this variant virus. Knowledge of this virus is still limited, and characterization of transmission properties of this resistant variant in in vitro and in vivo models is needed. Moreover, pandemic (H1N1) 2009 virus should be closely monitored for emergence of resistant variants.


A major challenge in disease ecology is to understand the role of individual variation of infection load on disease transmission dynamics and how this influences the evolution of resistance or tolerance mechanisms. Such information will improve our capacity to understand, predict, and mitigate pathogen-associated disease in all organisms. In many host-pathogen systems, particularly macroparasites and sexually transmitted diseases, it has been found that approximately 20% of the population is responsible for approximately 80% of the transmission events. Although host contact rates can account for some of this pattern, pathogen transmission dynamics also depend upon host infectiousness, an area that has received relatively little attention. Therefore, we conducted a meta-analysis of pathogen shedding rates of 24 host (avian) – pathogen (RNA-virus) studies, including 17 bird species and five important zoonotic viruses. We determined that viral count data followed the Weibull distribution, the mean Gini coefficient  was 0.687 (0.036 SEM), and that 22.0% (0.90 SEM) of the birds shed 80% of the virus across all studies, suggesting an adherence of viral shedding counts to the Pareto Principle. The relative position of a bird in a distribution of viral counts was affected by factors extrinsic to the host, such as exposure to corticosterone and to a lesser extent reduced food availability, but not to intrinsic host factors including age, sex, and migratory status. These data provide a quantitative view of heterogeneous virus shedding in birds that may be used to better parameterize epidemiological models and understand transmission dynamics. In the last century, there has been an unprecedented increase in the numbers of emerging infectious diseases (EIDs), which pose significant risks to wild and domestic animal and human populations The importance of transmission heterogeneity to the spread of disease is becoming increasingly recognized Quantitative information concerning the heterogeneity of viral load across infected individuals in a population would be the first step to establishing a link between supershedders and superspreaders. In this paper, we comprehensively evaluated the inequality of viral load and present a meta-analysis of 24 avian-virus experimental infection studies, which included 17 species of birds and five RNA viruses of three different families, Orthomyxoviridae, low and high pathogenic avian influenza virus (LP- and HPAIV); Flaviviridae, West Nile virus (WNV) and St. Louis encephalitis virus (SLEV); and Togaviridae, eastern equine encephalitis virus (EEEV) and western equine encephalitis virus (WEEV). RNA viruses in particular, are thought to be the causative agent for approximately 30% of the identified EIDs The role of birds in the ecology of a variety of diseases of public health concern is well documented The specific goals for this paper were to: (I) test the hypothesis that viral load in birds is consistently heterogeneous by (a) determining which data distribution function best fit viral load data  and (b) measuring the degree of inequality (via Gini coefficients) of viral load across individuals within an experimental study population; (II) evaluate factors associated with high viral shedding (supershedding); and, (III) indicate how our observations can be used to further understand and control avian-borne viral zoonotic diseases. We have found heterogeneous viral shedding patterns to be consistent across all 24 datasets evaluated. Based on our findings, we identify future research directions to understand the mechanisms driving these patterns as well as potential ways to link these findings with disease prevention and control strategies.Melopsittacus undulates) study). We use the term ‘shedding’ to reflect the transmissible fraction of virus burden, whether it is circulating virus in the blood (viremia) or actively deposited virus into the environment.We identified experimental infection studies from a variety of host-pathogen (bird-virus) systems that satisfied the following criteria: (a) individuals in each experiment were infected with a known dose of a viral pathogen , (b) the experimental birds were wild-type or wild-caught, so as to represent natural genetic variation, (c) pathogen load in the avian host was monitored for the entire infectious period, and (d) host pathogen load was measured at an anatomic site most relevant to transmission for a given host-pathogen system. The data represent a variety of published studies (mean n = 20.7 SEM 3.3) that were conducted by different laboratories and individuals  with greater than 30 subjects to evaluate the distribution of the data as described below. The remaining 20 datasets with fewer than 30 subjects were not used for distribution fitting. These data were then analyzed using R  for their probability distribution and degree of inequality as follows.n is the sample size, xi is a vector of data, and Determination of distribution functions and parameter estimates: The distribution functions for each of the four studies having greater than 30 subjects were determined by maximum likelihood estimation methods. Candidate probability distributions were Pareto, Weibull, Generalized Extreme Value (GEV), and exponential (EXP). These distributions each model long-tailed data via differing distribution functions and parameters. Using the ‘fitdistr’ function library ‘MASS’ in R Inequality of infection: We quantified the inequality of infection via the Gini coefficient using the ‘gini’ function in the ‘reldist’ package in R th, 80th and 90th virus shedding percentiles for each host-pathogen system.Lastly, to facilitate an intuitive understanding of these results, we determined 50th percentile, the latter were then classed as “supershedders”, see Discussion) and then tested for the effect of intrinsic  and extrinsic  factors on their status as a supershedder (yes or no) by logistic regression using SAS JMP 8.0. Odds ratios were estimated when a factor was determined to be statistically significant (P<0.05) for its effect on a bird’s classification as a supershedder.We next evaluated factors associated with the inequality of infection . The effect of anatomic site in which virus was measured, host species, virus species, or the number of individuals in the experimental infection study were evaluated for their effect on Gini  in SAS JMP 8.0 . Finally, we binned birds according to their relative virus shedding quantity (< or ≥80n ≥30) best fit the Weibull distribution . These data followed no other distribution that we evaluated . We subsequently estimated the scale  and shape  parameters for these and each of the 20 remaining datasets  (P>0.05).Across all 24 datasets and 20 unique avian-virus systems, we found that avian viral loads were not equally distributed across individuals and best fit a long-tailed data distribution function . The dish n <30) . The estth, 80th and 90th percentiles and found that a mean of 22.0% ±0.90 SEM of the birds accounted for 80% of the virus in an experimentally infected population  in a population account  for ∼80% of a material  pulation .2 = 3.43, df = 1, P = 0.064; Dumetella carolinensis), n = 17 and 59) No factor tested statistically significantly affected Gini . Howeverth percentile for a given study, placing them towards the left in the distribution of viral counts. No intrinsic factor evaluated  affected whether a bird was classed as a supershedder or not . Specifically, the exposure of WNV-infected chickens to corticosterone statistically significantly affected whether a bird was a supershedder  was classed as a supershedder . A normally fed bird was 4.38 times more likely to be a supershedder than a bird maintained on a food-restricted diet . However, while overall the birds in a state of reduced body condition contributed the least to the total group virus, the two highest shedders were in the lean and poor condition groups  and that viral ‘supershedders’ consistently emerge in an infected population, regardless of avian host or viral species. Quantitatively characterized heterogeneity in virus load provides enormous utility for developing epidemiological models that are a better representation of reality th percentile. Second, the Gini coefficient was greater than 0.600, indicative of a high degree of inequality or the 20/80 Rule (Pareto principle). Third, the estimated Weibull shape parameter was less than 1.0, indicating a long-tailed data distribution. Hence, regardless of how we analyze the data we find consistent support for a skewed distribution of the data  that can be described as following the Pareto Principle.Several lines of evidence in our study indicate that RNA-virus shedding in birds follows the Pareto Principle. First, it is visually evident that approximately 80% of the virus was shed by approximately 20% of the birds  and the in vitro or as a copy of viral RNA via RT-PCR and the plaque count or copy number are then tracked during the infection period. The Weibull distribution is described by scale (λ) and shape  was sensitive to mean parasite burden The mean shape parameter estimate  and dunlin  We found little evidence of intrinsic or extrinsic factors influencing Gini ; mean viThe consistent inequality of viral shedding  across identically exposed hosts is a key finding because it suggests that host physiological and/or virus replication mechanisms rather than differences in exposure are associated with the emergence of differentially infected individuals. For host-pathogen systems in which supershedding individuals may disproportionately contribute to epidemics, this information would be of great epidemiological advantage, especially when these individuals can be identified before or early in the course of their infection and prior to transmission. Problematically, such early detection of highly infectious hosts is currently not possible because the mechanisms driving supershedding are largely unknown and grossly understudied, especially in epidemiologically relevant wild hosts.nth percentile  of the transmission events 0) for a given host-pathogen system; however, this is not known for most wild organisms. In contrast to superspreading, there is no accepted definition of supershedding, which may be the result of inadequate knowledge about the role of host infectiousness on agent transmission and R0 in the wild E. coli in cattle. Specifically, they found that by identifying the 5% of the population with the highest mean infectiousness and targeting them for culling affected estimates of R0, more than any other factor evaluated.The transmission of an agent depends upon both contact rates between infected individuals or environments and uninfected individuals, and the ‘infectiousness’ of the infected individual. The latter depends on the amount of infectious pathogen an individual is circulating or sheds into the environment. Hence, superspreading is likely to depend on an individual’s propensity to ‘supershed’ infectious pathogen. Yet, quantitative definitions of both superspreading and supershedding have not been universally adopted. One definition that has been promulgated in recent studies th percentile. This definition takes into account the observed 20/80-distribution of virus shedding in the systems analyzed in this study, as well as practicalities of characterizing epidemiological parameters and host attributes potentially associated with these parameters in wild host-pathogen systems. If the goal is for enhanced understanding and mitigation of disease in animals, as well as humans, then determining the 20% of hosts responsible for 80% of the virus would be both highly advantageous as well as more cost-effective than the identification of the 1% of birds shedding the highest levels of virus For the purposes of our study, we defined supershedding as those individuals shedding at the 80The preemptive identification of viral supershedders and potential superspreaders is key for the early and rapid mitigation of a disease outbreak. Identifying host features associated with supershedding may facilitate more targeted research or disease control efforts, and this can only be accomplished with a thorough understanding of the mechanisms responsible for infection heterogeneity. The life cycle of a virus provides the basis for investigations of supershedding and includes (I) the initial exposure of a host, (II) entry into sites of virus replication within a host through barriers and receptors, (III) virus replication, and (IV) exposure of another host to the virus th percentile whereas no control birds shed at this level Extrinsic Factors: In two of the studies evaluated, we examined the impact of environmentally relevant factors on shedding inequality. Animal populations encounter conditions that can enhance the secretion of glucocorticoid hormones , a priori predictions. That is, mallards that experienced reduced food availability and body condition, shed lower levels of LPAIV than birds provided with ‘normal’ food allowances The second extrinsic factor that affected the position of a bird  in the CDF plot in our study was food availability, but in a manner contrary to our th percentile (“supershedders”)  . A lack Virulence, an emergent property, is a consequence of coevolutionary processes between hosts and parasites in the context of constantly changing environments In the current study, viral strain and often exposure concentration were held constant within an experiment, meaning that viral evolution would likely be dependent more upon the selective pressures imposed by the host environment than an initial variation of viral traits th percentile, hatchery raised mallards infected with AIV and provided with a “normal” diet did not uniformly shed at a different rate than wild-collected mallards exposed to the same experimental conditions  In cases when differential pathogen shedding influences epidemic growth rates, examine the role of factors extrinsic and intrinsic to the host on the development of differential pathogen load, with the goal of early detection of supershedders. (4) Examine whether genetic or physiologic biomarkers exist for supershedders of a pathogen, which could be used for early detection and identification of highly infectious individuals. (5) Collect pathogen load data in diverse systems including agricultural animals, wildlife, and humans to investigate environmental correlates with the presence of supershedding individuals. The coupling of experimental investigations of the determinants of supershedding and transmissions dynamics with epidemiological modeling, could lead to the identification of a critical missing component in the mitigation of disease in both animal and human populations.Given that we have specifically demonstrated that virus shedding is not equally distributed across equally  infected birds and that we know that superspreading events can depend upon either or both contact rates and host infectiousness, we suggest the following research directions. (1) Understand the role of supershedding individuals on the potentiation of superspreading events for a given disease system including comparisons between vector-borne and directly transmitted viruses. (2) For a given disease system, determine whether heterogeneous contact rates and heterogeneous shedding independently, additively, or synergistically affect epidemiological rates such as R


Mastomys natalensis), Morogoro virus, related to Mopeia virus. Only a small percentage of mice carry Morogoro virus, although a large proportion shows specific antibodies.A serosurvey involving 2,520 small mammals from Tanzania identified a hot spot of arenavirus circulation in Morogoro. Molecular screening detected a new arenavirus in Natal multimammate mice ( Arenaviruses are segmented negative-strand RNA viruses. Their natural hosts are various rodent species. The virus family comprises several human pathogens causing hemorrhagic fever, namely Machupo, Guanarito, Junin, Sabia, and Chapare viruses in South America, and Lassa and Lujo viruses in Africa (Arvicanthis spp. rodents from the Iringa region (20%) and in Natal multimammate mice  from Arusha (18%) and Morogoro (17%) .During 1985 through 1989, a total of 2,520 small mammals were live-trapped in different regions of Tanzania. After species determination, they were measured and bled by orbital puncture. Serum samples were tested by indirect immunofluorescent antibody (IFA) assay (ro (17%) , which sM. natalensis mice were trapped in a mosaic of maize fields and fallow grassland at the university campus in the city of Morogoro  to identify the virus. The animal voucher specimens were deposited at the Royal Museum of Central Africa, Tervuren, Belgium. RNA was prepared from 10 μL of rodent serum by using the QIAamp Viral RNA kit , and screening was performed by using a pan–Old World arenavirus reverse transcription–PCR (RT-PCR) specific for the large (L) gene  gene , and seqMastomys natalensis, EU914105–06.)For sequencing, the isolate was propagated in T75 flasks, virus particles in supernatant were pelleted by ultracentrifugation, and RNA was isolated by using the QIAamp Viral RNA kit (QIAGEN). The entire 3.5-kb small (S) RNA segment was amplified by RT-PCR as described previously (Full-length amino acid sequences of glycoprotein precursor (GPC), nucleoprotein (NP), and L protein of Morogoro virus were aligned with published Old World arenavirus sequences and pairwise p distances were calculated. Morogoro virus showed genetic similarity to strains of Mopeia virus that were circulating in Mozambique .Although the distances between Morogoro and Mopeia virus in the amino acid sequence of GPC (12%), NP (12%–13%), and L gene (26%) were higher than intraspecies differences among known African arenaviruses , they did not reach the level of interspecies distances   was homogenized by using a bead mill. Cell debris was pelleted by centrifugation, and RNA was isolated from the homogenate with the RNeasy Mini kit (QIAGEN). Testing by L gene RT-PCR (n of ≈4% . PCR fran of ≈4% .http://beast.bio.ed.ac.uk) (http://tree.bio.ed.ac.uk/software/tracer). Based on GTR and HYK model, 3.2 × 10–3 and 3.4 × 10–3 substitutions per site and year , respectively, were calculated.The availability of Morogoro virus L gene sequences from 2004 and 2007, originating from the same host population (trapping sites <1 km apart), provided us with the opportunity to estimate the molecular clock rate for this virus. Phylogenetic reconstruction was performed with the BEAST version 1.4.8 package (M. natalensis mice carry Morogoro virus, and a large proportion shows specific antibodies, which indicates that most animals clear the virus during life. Viruses and antibodies, which are presumbably directed to nucleocapsid proteins, also co-circulate, as seen in hantavirus infection in rodents (M. natalensis mice (A serologic survey in small mammals from Tanzania identified a hot spot of arenavirus circulation in Morogoro in the late 1980s. This work is being published now because early attempts to substantiate the existence of the virus failed. The identification of the virus was facilitated by a recently developed pan–Old World arenavirus PCR (–3 for Morogoro virus L gene is in agreement with that of other RNA viruses (The clock rate estimate of 3 × 10The pathogenicity of Morogoro virus for humans is not known, though its phylogenetic clustering with African arenaviruses that are not linked with human disease (


Wild birds, especially those in wetlands and aquatic environments, are considered to be natural reservoirs of avian influenza viruses. It is accepted that water is an important component in the transmission cycle of avian influenza virus. Monitoring the water at aggregation and breeding sites of migratory waterfowl, mainly wetland, is very important for early detection of avian influenza virus. The epidemiology investigation of avian influenza virus was performed in Dongting lake wetland which is an international important wetland.An H10N8 influenza virus was isolated from Dongting Lake wetland in 2007. Phylogenetic analysis indicated that the virus was generated by multiple gene segment reassortment. The isolate was lowly pathogenic for chickens. However, it replicated efficiently in the mouse lung without prior adaptation, and the virulence to mice increased rapidly during adaptation in mouse lung. Sequence analysis of the genome of viruses from different passages showed that multiple amino acid changes were involved in the adaptation of the isolates to mice.The water might be an important component in the transmission cycle of avian influenza virus, and other subtypes of avian influenza viruses  might evolve to pose a potential threat to mammals and even humans. All 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes of influenza A virus have been isolated from wild birds . TherefoDongting Lake wetland is an important habitat and over-wintering area for East Asian migratory birds, and is located at 28°30'-30°20' N and 111°40'-113°40' E in the Northeastern part of Hunan Province, China. In 2007, an influenza virus A/environment/Dongting Lake/Hunan/3-9/07 (H10N8) was isolated from water from Dongting Lake wetland. The whole genome of the isolated virus was sequenced, the phylogenetic trees of each gene segment were generated, and the pathogenicity of the strain for mice and SPF White Leghorn Chickens was studied. To study further its potential pathogenicity for mammals, the virus was passaged in mouse lung, and the pathogenicity and corresponding amino acid variations of the mouse-lung-adapted virus from passages 2, 4 and 6  were compared with those of wild-type virus (P0).An H10N8 influenza A virus was isolated from water samples from Dongting Lake wetland, and named as A/environment/Dongting Lake/Hunan/3-9/2007 (H10N8) (environment/DT/Hunan/3-9/07). The whole genome of the isolated virus was sequenced to understand the genetic character of the virus.BLAST analysis of the eight gene segments of environment/DT/Hunan/3-9/07 revealed the presence of an HA gene that was closely related to that of A/duck/Mongolia/149/03 (H10N5), with a nucleotide sequence identity of 96% and amino acid sequence identity of 97% , and another 8 chickens were inoculated intranasally with virus in a volume of 0.1 ml (106.0EID50), and observed for clinical signs of disease and mortality for 14 days. The oropharyngeal and cloacal swabs of chickens were collected on days 3, 5 and 7 post inoculation (p.i.). for virus titration. None of the chickens challenged by intravenous or intranasal virus showed any clinical signs of disease within 14 days p.i., and none died during the observation period. These results suggested that the H10N8 strain was a low or non-pathogenic virus. Sera were harvested from the chickens at 21 days p.i. and seroconversion was confirmed by hemagglutination inhibition (HI) test. All the inoculated birds were seroconverted, although the HI antibody titers remained low throughout the experimental period , the virus caused fatal infection in mice. Mice inoculated with P2 virus showed serious clinical signs of disease such as ruffled fur, less movement and body weight loss .It was found that, during passage in the mouse lung from P0 to P6, 22 amino acid substitutions appeared, i.e. sites 207, 616 and 627 of PB2 gene; sites 247 and 611 of PA gene; sites 94, 244, 252, 386 and 430 of HA gene; site 479 of NP gene; sites 21, 32, 286, 330 and 385 of NA gene; sites 53 and 192 of M1 gene; site 82 of M2 gene; and sites 54, 89 and 155 of NS1 gene  BALB/c mice  were purchased from Hubei Research Center of Laboratory Animal, China. The SPF white Leghorn chickens (aged 6 weeks old) were purchased from Beijing Merial Vital Laboratory Animal Technology CO., LTD. Mice and Chickens were all bred in the Animal Resource Center at the Wuhan Institute of Virology, Chinese Academy of Sciences, maintained in specific-pathogen-free conditions prior to infection, and cared for under MOST (Ministry of Science and Technology of the People's Republic of China) guidelines for laboratory animals. All experiments involved in animals have been approved by Animal Care Committee of Wuhan Institute of Virology, Chinese Academy of Sciences.In October 2007, 95 water samples from areas near the habitat of migratory birds in East Dongting Lake, Yueyang City, Hunan Province were collected by using sterilized 200-ml screw-cap plastic vials. A 200-ml water sample was collected at each sampling site, stored in a portable refrigerator, sent to our laboratory, and stored at -80°C until assayed.g for 30 min[6 U/l penicillin, 2 × 106 U/l amphotericin B, 250 mg/l kitasamycin, 0.5 × 106 U/l nystatin, and 60 mg/l ofloxacin HCl. Then, 0.5 ml of the re-suspended mixture was inoculated into the allantoic cavities of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs and incubated at 37°C for 72 h. The allantoic fluid with hemagglutination titers were harvested and confirmed as influenza A virus stock by RT-PCR, using NP-gene-specific primers and universal primers for the M gene of influenza A virus, as described previously[Seventy milliliters of each water sample was transferred into a sterilized 80-ml polyethylene plastic tube with a screw-cap and round bottom, under aseptic conditions. Polyethylene glycol 6000, sodium chloride and bovine serum albumin (BSA) were added to final concentrations of 8%, 3% and 0.1%, respectively, mixed gently, set on ice for 8-12 h during which the tube was inverted every 2 h to mix the contents, and centrifuged at 4°C, 10,000×or 30 min. The supreviously,28. The The viruses were clonally purified by plaque isolation in MDCK monolayers, followed by stock preparation as described previously ,13.Total RNA from the virus genome was extracted from the prepared virus stock by lysing with Trizol LS reagent (Life Technologies) and reverse-transcribed into single-stranded DNA with M-MuLV reverse transcriptase (New England Biolabs). All segments were amplified with Phusion™ High-Fidelity PCR Kit (New England Biolabs). The PCR products were purified with the Cycle-pure Kit and Gel Extraction Kit (OMEGA), and the fragments were cloned into pGEM-T easy vector and sequenced by the dideoxy method with an ABI 3730 DNA sequencer (Applied Biosystems). Three clones of each gene were selected for repeated sequencing to confirm that the sequence data obtained on the two occasions were identical. Data wePhylogenic trees were generated with neighbor-joining bootstrap analysis (1000 replicates) using the Tamura-Nei algorithm in MEGA version 3.1 .6.3 EID50). Meanwhile, another 8 chickens aged 6 weeks were inoculated intranasally with 0.1 ml 106.0 EID50 virus. The inoculated chickens were observed for 14 days for mortality and clinical signs of disease. Tracheal and cloacal swabs were collected on days 3, 5 and 7 post inoculation (p.i.) for virus titration[50 was calculated by the Reed and Muench method. Sera were harvested from the inoculated chickens on day 21 p.i. for seroconversion confirmation by hemagglutination inhibition (HI) assays with 0.5% chicken erythrocytes according to the recommendation of OIE.Eight SPF White Leghorn Chickens aged 6 weeks were intravenously inoculated with 0.2 ml of a 1:10 dilution of bacteria-free allantoic fluid that contained virus . Five mice in each group were dissected on days 3 and 5 p.i., and their lungs, spleens, kidneys and brains were taken out under aseptic conditions, weighed and homogenized with 1 ml PBS that had been pre-cooled in ice. Tissue homogenates were centrifuged at 4°C,4,000×g for 10 min to remove any tissue fragments, and used to determine virus titer[50) of the virus was determined by inoculating intranasally 5 groups of mice (n = 5 mice each) with 10-fold serial dilutions of the virus in a volume of 50 μl. The MLD50 was calculated by the method of Reed and Muench.The BALB/c mice aged 6 weeks were divided into 4 groups of 16 each, anesthetized, and inoculated intranasally with P0 (wild-type), P2, P4 and P6 virus in a volume of 50 μl  have been submitted to GenBank and are available under accession numbers The authors declare that they have no competing interests.HBZ carried out most of the experiments and wrote the manuscript. BX, QJC and JJC did part of the experiment. ZC was the main designer of the experiment and revised the manuscript. All authors read and approved the final manuscript.


In recent years, our understanding of the role of natural killer (NK) cells in the response to viral infection has grown rapidly. Not only do we realize viruses have many immune-evasion strategies to escape NK cell responses, but that stimulation of NK cell subsets during an antiviral response occurs through receptors seemingly geared directly at viral products and that NK cells can provide a memory response to viral pathogens. Tremendous knowledge has been gained in this area through the study of herpes viruses, but appreciation for the significance of NK cells in the response to other types of viral infections is growing. The function of NK cells in defense against poxviruses has emerged over several decades beginning with the early seminal studies showing the role of NK cells and the NK gene complex in susceptibility of mouse strains to ectromelia, a poxvirus pathogen of mice. More recently, greater understanding has emerged of the molecular details of the response. Given that human diseases caused by poxviruses can be as lethal as smallpox or as benign as Molluscum contagiosum, and that vaccinia virus, the prototypic member of the pox family, persists as a mainstay of vaccine design and has potential as an oncolytic virus for tumor therapy, further research in this area remains important. This review focuses on recent advances in understanding the role of NK cells in the immune response to poxviruses, the receptors involved in activation of NK cells during poxvirus infection, and the viral evasion strategies poxviruses employ to avoid the NK response. Natural killer (NK) cells are generally part of the first line of defense to viral infection, but their relative importance to the immune response can vary depending on the virus . NK cellSuggestive of their ancient origin, poxviruses infect a wide variety of forms of life with families of poxviruses that infect invertebrates, reptiles, birds, and mammals. Poxviruses that infect mammals are further divided into a number of subgroups including the well-studied orthopox genus. The orthopox genus includes many different viruses that are both medically relevant and well studied as models for pathogenesis such as: ectromelia virus, a natural pathogen of mice; variola virus, the causative agent of smallpox; cowpox, a virus that is maintained in rodents but able to infect a broad range of mammals; and vaccinia virus, a virus likely derived from an extinct equine virus. Vaccinia virus was used as the vaccine successfully to eradicate variola virus and remains under investigation as a general vaccine vector as well as an oncolytic virus. Orthopox viruses cause a wide range of diseases and even a single type of virus will have a range of clinical course in different individuals. For example, while variola virus caused only a mild and localized disease in some individuals, many succumbed to severe disease with very high mortality rates especially for some strains of the virus  proteins. However, poxviruses do devote a considerable proportion (~50%) of the their rather large genomes (~200 genes) manipulating the hosts’ innate and adaptive immune defenses , NK cells are found in increased numbers in the popliteal lymph node 2 days after infection, while the peak in spleen and liver occurs at 6 days . NK cell6 pfu). Older studies show that activated NK cells accumulate in the peritoneal cavity in response chemokines following an intraperitoneal inoculation with vaccinia virus .Precisely how NK cells become activated during poxvirus infection is not entirely clear and likely has features that are dependent on the virus in question. Given the key role of NK cells in limiting viral replication early and that the control requires both IFN-γ and perforin mediated mechanisms , severalin vivo does increase viral dissemination. However, the effect of NKG2D blockade is not as complete as removing NK cells with depleting antibodies  inhibitors  was initially thought to be negligible because similar resistance to the virus was found when the H-2ckground . Subsequb itself . It is qb itself . On the b itself . Ly49h-/Figures 2). Highlighting these is the elucidation of CD94/NKG2E as a key stimulatory receptor required for resistance to ectromelia virus likely in conjunction with NKG2D. Although CD94/NKG2A appears to be necessary for protection from ectromelia virus in C57BL/6 mice, other susceptible strains such as BALB/c express CD94 but are highly susceptible to ectromelia indicating CD94/NKG2E is necessary but not sufficient for resistance. Several interesting questions remain in regard to CD94/NKG2E activation such as what causes the increase in Qa-1, if the peptide loaded into Qa-1 plays a role, and if this receptor plays a role in any other species. Given the homology with NKG2A, it may also be interesting to better understand the mechanism of vaccinia virus A40R that manifests its phenotype only upon dermal infection. Also, since C57BL/6 mice require TLR2 to respond to vaccinia virus, but CD57BL/6 TLR2-/- mice survive ectromelia infection , is coincidentally located at 19q13.4, near the region that also encodes the leukocyte receptor complex containing the KIRs ,b. A study just published demonstrates that when used as oncolytic virus, poxviruses can prevent post-surgical metastasis by increasing NK activity . TogetheThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To the Editor: Hepatitis E virus (HEV) is endemic to India , which has sensitivity >98%, was used to test serum samples for HEV IgM; real-time reverse transcription PCR (rRT-PCR) was used to test serum and stool samples for viral RNA  was used to align sequences. MEGA5  men had a febrile illness; 9 illnesses were accompanied by digestive signs or symptoms, such as nausea and vomiting . Only thIllnesses in infected and noninfected men did not differ, and, except for the notified case, we cannot say with certainty that the illnesses were caused by HEV. We found IgG against HEV in 14 (34%) uninfected men, which is close to the prevalence for the general population in India (HEV subtype 1a isolates from the notified case-patient (serum-derived isolate) and a co-worker (stool-derived isolate) were sequenced  and found to be 100% similar and to share 97%– 99% similarity with other HEV strains in India and Nepal . The seq>3 weeks for HEV  (Characteristics of the population studied and association with hepatitis E virus (HEV) infection, and a phylogenetic tree constructed with HEV sequences for virus strains from participant from India, persons in Central African Republic with autochthonous infection, and reference strains.