Influenza A virus is one of world’s major uncontrolled pathogen, causing seasonal epidemic as well as global pandemic. This was evidenced by recent emergence and continued prevalent 2009 swine origin pandemic H1N1 Influenza A virus, provoking first true pandemic in the past 40 years. In the course of its evolution, the virus acquired many mutations and multiple unidentified molecular determinants are likely responsible for the ability of the 2009 H1N1 virus to cause increased disease severity in humans. Availability of limited data on complete genome hampers the continuous monitoring of this type of events. Outbreaks with considerable morbidity and mortality have been reported from all parts of the country.Considering a large number of clinical cases of infection complete genome based sequence characterization of Indian H1N1pdm virus and their phylogenetic analysis with respect to circulating global viruses was undertaken, to reveal the phylodynamic pattern of H1N1pdm virus in India from 2009–2011. The Clade VII was observed as a major circulating clade in phylogenetic analysis. Selection pressure analysis revealed 18 positively selected sites in major surface proteins of H1N1pdm virus.This study clearly revealed that clade VII has been identified as recent circulating clade in India as well globally. Few clade VII specific well identified markers undergone positive selection during virus evolution. Continuous monitoring of the H1N1pdm virus is warranted to track of the virus evolution and further transmission. This study will serve as a baseline data for future surveillance and also for development of suitable therapeutics. Orthomyxoviridae. Based on the antigenicity, virus may be classified into 16 Hemagglutinin (H1–H16) and 9 Neuraminidase (N1–N9) subtypes. Influenza A virus genome is composed of eight segments of single-stranded, negative-sense RNA and each of which encodes one or two proteins. The HA protein is critical for binding to cellular receptors and fusion of the viral and endosomal membranes. Replication and transcription of viral RNAs (vRNAs) are carried out by three polymerase subunits PB2, PB1, and PA, and the nucleoprotein (NP). Newly synthesized viral ribonucleoprotein complexes are exported from the nucleus to the cytoplasm by the nuclear export protein  and the matrix protein M1, and are assembled into virions at the plasma membrane. NA protein cleaves sialic acid residues on the host cell glycoproteins and glycolipids to which the HA proteins of newly assembled virions bind and, therefore plays an important role in the release of newly formed virions from the host cell membrane Influenza A virus is known to cause an acute respiratory disease with a history of causing severe pandemics including the recent one by novel swine origin Influenza A virus (S-OIV). The property of virus subtype to mutate into variety of strains with differing pathogenic profile, eventually resulted in achieving higher fitness in a brief period. Influenza A virus is a member of family Several reports described both emergence and pandemic potential of the virus in the perspective of earlier pandemic influenza viruses of 1918 (H1N1), 1957 (H2N2) and 1968 (H3N2) through comparison of the available genetic sequence data The major symptoms of the disease is characterized by the sudden onset of high fever, chills, coughing, sore throat, muscle pain, severe headache, malaise, and inflammation of the upper respiratory tract and trachea, with general discomfort, but it rarely induces severe inflammatory lung diseases, including pneumonic involvement due to host innate and acquired immunity. Swine origin pandemic human influenza A virus (H1N1pdm) has spread rapidly around the world since its initial documentation in April 2009. According to last update (29 Jan 2010- update 85) of WHO in pandemic period H1N1pdm had spread to 209 countries and overseas territories, with 14711 deaths since the first reports of the virus in human in April 2009. In India the H1N1pdm virus is circulating through its emergence continuously and viral cases are being reported from different parts of the country in post pandemic phase Understanding the virus evolution within India in relation to global diversification of the virus is also essential. So far, not much data is available on complete genome characterization of Indian H1N1pdm virus. The circumstances surrounding the emergence of this pathogen, and the factors that facilitated the initial cross-species transmission, are still not fully understood. It became apparent in the early days of the outbreak that the virus can be directly transmitted between humans. Among the various efforts made to evaluate, diagnose and implement the measures against the spread of virus, is the timely release of the genomic sequences from different viral isolates 35 patients (WHO category C cases) were confirmed positive by CDC real time RT-PCR with positivity of 29.16%. The youngest case was a 6 months old female child. Monthly sample analysis profile revealed that 92.5% of the samples pertained to the period September-December 2010–2011, and the rest 7.5% of cases reported besides this period. 47.5% cases were seen amongst the age group of 20 to 39 years, while 15.83% cases were seen amongst the age group of 5–19 years. The median age of the samples investigated was 30 years (range 6 months- 76 years). 6.66% of the patients were under age 5 and 10.83% were more than 54 years old. The female/male ratio for H1N1pdm in different age groups were significantly greater than 1. No patient was previously vaccinated, however oseltamivir was started after 5 days in 30% of the cases. An overall case fatality rate was 8.33% with 10 deaths. Maximum deaths were seen in younger age group (7–25 years) with increased case fatality rate of 15% in 2011. Death in complicated cases occurred between 24–48 hours of report to hospitals. The clinical history revealed that all the patients had suffered from fever (>38.0°C). Other prominent clinical symptoms include fever  (80%), cough (42%), sore throat (38%), nasal catarrh (75%) and shortness of breath (66%). Monthly and age wise distribution of suspected patients is summarized in Out of 120 suspected samples, 35 (29.16%) were positive for pandemic Influenza A H1N1 and 7 (5.83%) were positive for Influenza A . The cases of H1N1pdm started rising from September 2010 with maximum number of cases (n = 44). All the samples were diagnosed by WHO approved CDC Real time RT-PCR using 4 sets of primer and probes. Samples found positive for all the four probes viz. Influenza A, Swine Influenza A, Swine H1, RNase P  were declared positive for H1N1pdm virus. Each lot of samples were tested with a positive confirmed H1N1pdm cell culture RNA as positive control and healthy throat swab sample RNA as negative control. Detailed features including clinical presentations of H1N1pdm positive samples were summarized in Three selected positive samples were attempted for the H1N1pdm virus isolation in MDCK cells through three blind passages. Initially, H1N1pdm virus infection in MDCK cells was analysed microscopically for the appearance of prominent cyotopathic effects  till 48–72 hpi . InfecteA total of 4 representative H1N1pdm viruses comprising two 2010 isolates  and two 2011 isolates  of Gwalior were selected for complete genome amplification. These samples at passage level three in MDCK cells were used for the purpose and were subjected to complete genome amplification. The editing and alignment of sequences of overlapping fragments led to sequence information of complete genome. Complete genome (concatenated eight gene segments) of isolates deciphered was 13158nt. The sequences  for all the four isolates were deposited in GenBank with following accession numbers JF265678, JF265677, JF764085, JF764086, JF265676, JF265675, JF293316, JF293315, JF265674, JF265673, JF265672, JF265671, JF764082, JF510037, JF764083, JF764084, JQ 319657-58, JX 262203-04, JX 262207-08, JX262205-06, JX 262209-10, JX 262201-02, JX 262211-12, JX 262213-14.The genome sequences of representative Influenza A (H1N1pdm) viruses of diverse geographical origins were retrieved from NCBI GenBank database from the period of 2009–2012 . ComparaExtensive phylogenetic analysis based on concatenated whole genome sequences  and full HA gene  of representative H1N1pdm viruses sampled between 2009–2012 from different geographical regions along with the Indian isolates revealed seven distinct clades  . Both tComparison of individual gene segment at protein level with respect to A/California/04/2009 (H1N1pdm prototype strain) and A/India/Pune/NIV6447/2009 (previously sequenced Indian strain) revealed a total of 73 substitutions scattered throughout the eight gene segments in four Indian viruses sequenced in this study. The sequence alignment revealed amino acid replacement throughout the aligned region. The 47 major/important non-conservative and clade specific amino acid substitutions among H1N1pdm virus (sequenced in this study) vis-a-vis prototype California/04/2009 and A/India/pune/NIV6447/2009 are shown in Selection pressure analysis of HA, NA and MP gene of 72 global H1N1pdm virus strains revealed 18 positively selected sites. Integrated analysis was performed for differential selection pressure acting on HA (566 codons), NA (469 codons), M1 (252 codons) and M2 (97 codons) proteins. Positive selection on HA gene was stronger than NA, M1 and M2 protein gene. In total 11 HA, 3 NA, 2 M1 and 2 M2 sites were found under positive selection by at least two methods . Out of Transmission of pandemic Influenza virus is persisting in many continents but current activity levels are low in Asia. Recent peaks in the activity were noted during early 2010 in northern India, Nepal and Sri Lanka. Influenza activity remained stable but elevated in western India, continued to decline substantially in Northern India, and remained low overall in Southern and Eastern India In this particular study, the main focus was complete genome characterization of the circulating isolates of northern India  and to decipher conservative and non conservative substitutions, its comparative analysis with respect to other Indian and global circulating H1N1pdm isolates. The continued circulation of virus in particular region from 2009-till date is also a serious concern and required in depth investigation. With the determined objective of molecular investigation of circulating H1N1pdm virus, Influenza like illness (ILI) in suspected clinical samples from Gwalior, India during 2010–2011 were investigated. The clinical picture of the patients revealed the same pattern as was reported in 2009 In this study four Indian isolates that are confirmed by virus specific CPE, HA, IFT as well as CDC Real time RT-PCR were selected for complete genome characterization. The nucleotide sequence analysis revealed that there is no significant difference among viruses recovered from two different places and of different years from India. Diversity of the Indian isolates at the amino acid level with respect to the prototype strain and within the Indian isolates was found to be maximum in the HA and NP gene. Substitution S220T (HA) specific to clade VII isolates was adequate to lineate the isolates in HA-based phylogeny. Most of the amino acid changes were conservative, involving interchanges of amino acids having same physicochemical properties. However, few major non-conservative changes between Indian isolates were also observed. Compared to the prototype strain, glutamic acid was replaced by a strongly basic amino acid lysine at position 391 (HA) among the four Indian H1N1pdm virus and at the position 71 (NS1) in one Indian H1N1pdm virus sequenced in this study. Two important non conservative substitutions involving acidic aspartic acid to basic histidine at position 441(PB2) in two Indian H1N1pdm virus and cyclic proline to acyclic serine at position 100(HA) among the four Indian H1N1pdm virus were also recorded. Similar non conservative substitutions involving shift in amino acids were also recorded in other gene segments. However, the significance of these substitutions need to be addressed.To identify genetic lineage of H1N1pdm virus, phylogenetic analysis was conducted for concatenated whole genome sequences retrieved from GenBank from 2009–2012 including all the available H1N1pdm whole genome from India sequenced till date. Whole genome and full HA based phylogenetic analysis revealed existing seven discrete clades of H1N1pdm virus circulating globally. Both the trees based on genome information comprised of all representative H1N1pdm clades from diverse geographical origin which included maximum number of representative H1N1pdm from all the affected areas. Both the trees yielded similar topologies, with characteristic distribution of H1N1pdm isolates into seven distinct clades. Maximum numbers of isolates were grouped into clade VII. The clade I included prototype California/04 and California/07 virus isolated first during H1N1pdm Influenza viruses comprise of segmented viral genome, and are more prone to genetic reassortment during mixed infections. Hence the circulating H1N1pdm strains also evolve and may favourably be selected with higher fitness at a particular time point. It is most likely that the H1N1pdm strains were also undergone similar evolutionary process and the viruses of higher fitness were favourably selected over time. The selection pressure analysis revealed 18 positively selected sites in major surface proteins of Influenza A (H1N1pdm) virus i.e. HA, NA and matrix proteins. Since these proteins plays crucial role in the attachment, assembly, release of the virus, these substitutions might have played important role in making these isolates more transmissible. Differential selection analysis also supported the pandemic 2009 strains being subject to distinctive selection compared to their progenitors The present study is the first systematic study carried out to characterize the true genetic nature of recently circulating Indian H1N1pdm virus in post-pandemic phase. This study clearly indicates that the cosmopolitan clade VII is predominant in India. Few reported Clade VII markers revealed in this study indicates that the clade is undergone positive selection during virus evolution since last 3 years and a shift to clade VII in Indian isolates was observed from other circulating clades during 2009–2012. The complete genome information of recent H1N1pdm Indian virus isolate elucidated for the first time in this study will serve in future epidemiological surveillance in Indian subcontinent and abroad.A total of 120 acute phase throat/nasopharyngeal swab samples suspected for H1N1pdm virus, with Influenza A like illness between 3–7 days of onset of fever  were referred from sentinel hospitals in Gwalior, India for the laboratory investigation of H1N1pdm outbreak during 2010 and 2011. Throat/nasopharyngeal swab samples were received in viral transport medium (Himedia) at appropriate cold temperature (4°C) and triple packaging system. All the samples were processed in the High Containment Facility (a biosafety level −3 laboratory) at DRDE, Gwalior. A total of four Indian isolates  were selected for the complete genome sequence and phylogenetic analysis in this study. Out of positive samples, three viruses isolated from Gwalior, India  and one virus isolated by Prof. V. Ravi, at NIMHANS, Bangalore (A/India/Blore/2010) were included for complete genome characterization.Viral RNA was extracted from 140 µl of clinical sample and cell culture supernatant (Isolates) by using QIAamp viral RNA mini kit  in accordance with the manufacturer’s instructions. Finally, RNA was eluted in 50 µl of elution buffer and stored at −80°C until use.TMIII Platinum® one step quantitative kit. The amplification was carried out in a 25 µl reaction volume according to the CDC instruction and standard thermal profile for sample screening The CDC Real-time RT-PCR assay was used for novel swine flu virus identification in MX 3000P quantitative PCR system . The assay is based on Taqman chemistry including a panel of oligonucleotide primers and dual labeled hydrolysis probe sets  employing Invitrogen SuperScript2 atmosphere. The clinical samples  obtained from patients were inoculated in MDCK cell lines at 90% confluency for virus isolation as per standard protocol All the clinical samples were processed in Biosafety Level −3 Laboratory. Madin Darby Canine Kidney (MDCK) cells purchased from NCCS, Pune were maintained in Modified Eagle’s Medium (MEM)  supplemented with 5% fetal bovine serum (FBS) (Sigma-Aldrich) at 37°C in a humidified 5% COHemagglutination (HA) test was performed using guinea pig RBC following standard protocol 2 (3 mM), 0.5 µl dNTP (200 µM each), 0.5 Reverse Transcriptase (0.4 units/µl), 0.5 µl RNAse inhibitor (0.4 units/µl), 0.5 µl TaqDNA polymerase (0.05 units/µl), 0.25 µl of respective forward and reverse primers and 13 µl of molecular biology grade water. The One-step RT-PCR was carried out using Enhanced Avian HS RT-PCR kit . The PCR amplification was carried out in a final volume of 25 µl in a thermal cycler . The thermal profile comprised of reverse transcription at 48°C for 45 min, initial denaturation at 95°C for 2 min followed by 35 cycles at 95°C for 1 min, annealing at 56–65°C for 1 min, extension at 72°C for 2 min and final extension at 72°C for 10 min. The PCR products were gel purified from 1% agarose gel using the QIAquick gel extraction kit  and used as template in sequencing reactions.One step RT-PCR was carried out to amplify all the eight segments using the recommended WHO-CDC whole genome primers Taq DNA polymerase, dNTPs and four dye-labelled dideoxy nucleotide terminators (ddNTPs) and 25 ng of purified PCR product, and 3.2 pmol of either sense or antisense primer. Cycle sequencing parameters were as follows: 25 cycles of 96°C for 5 sec, 50°C for 15 sec, and 60°C for 4 min). The reaction mixture was column purified and the DNA was dried in vacuum. The DNA pellet was resuspended in 15 µl of hidiformamide, heated at 95°C for 5 min before loaded on the ABI 310 automated DNA sequencer .Double pass sequencing was carried out employing big dye terminator cycle sequencing ready reaction kit  on an ABI 310 sequencer. Briefly, each sequencing reaction was carried out in a final volume of 10 µl by mixing the Big Dye terminator mix containing the thermostable AmpliSeqScape  and EditSeq and MegAlign modules of Lasergene 5 software package . Multiple sequence alignment was carried out employing MUSCLE EditSeq module of Lasergene 5 software package . The percent nucleotide identity and percent amino acid identity values were calculated as pairwise p-distances. Extensive phylogenetic analysis based on full HA gene (1701 nt) and complete genome (13158nt: concatenated eight segments) were carried out by including 45 and 65 globally diverse H1N1pdm sequences  was found to be the best-fit model for our dataset. Convergence was assessed using mean SD in partition frequency values by using a threshold of 0.01.The nucleotide sequences were retrieved, edited and analysed using the equences  respectihttp://www.datamonkey.org/) Selection pressure analysis acting on the codons of surface proteins i.e. hemagglutinin (HA), neuraminidase (NA) and matrix protein (MP) of H1N1pdm virus was carried out using HyPhy open-source software package available under the datamonkey web-server Click here for additional data file.Table S2Gene bank accession numbers used in selection pressure analysis of HA, NA and Matrix protein gene.(DOC)Click here for additional data file.Table S3Selection pressure analysis of HA protein (566 codons); NA protein (469 codons), Protein of Indian H1N1pdm virus using SLAC, FEL,REL,MEME and FUBAR methods. (www.datamonkey.org).(DOC)Click here for additional data file.


Low pathogenicity avian influenza (LPAI) viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI) virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1) showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA) gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5%) of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing. Avian influenza (AI) viruses are divided into subtypes on the bases of the antigenic properties of their two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). To date, a total of seventeen HA and ten NA subtypes are known, and, with the exception of recently identified H17N10 subtype which was isolated from bats in vitro and in vivo procedures; these include continued passage of a LP virus in tissue culture in vivo in turkeys to generate HPAI viruses during infection with a LP chicken-origin virus. We have previously reported on in vivo infection of turkeys with a LPAI virus which resulted in severe disease signs and death The evolution of LP to HP virus pathotypes of H7 and H5 subtypes has been reported in field and experimental infections in chickens and turkeys To investigate the possibility of virus pathotype evolution over the course of the infection, buccal and cloacal samples collected over the course of infection and tissues harvested from humanely killed birds with clinical signs were analysed for the presence of a molecular signature of HPAI virus. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplicons of the HA gene were analysed by Sanger and Next Generation Sequencing (NGS), and through a sensitive RT-PCR assay.This study was carried out according to the guidance and regulations of the UK Home Office with appropriate personal and project licences (licence number 70/6201). As part of this process the work has undergone scrutiny and approval by the ethics committee at the AHVLA-Weybridge.50) according to standard protocols The LPAI H7N1 virus strain A/chicken/Italy/1279/99  was isolated during an outbreak of avian influenza in Italy in 1999–2000 . Virus stocks were grown in 9-day-old specified pathogen free (SPF) embryonated fowls’ eggs , and virus titres were calculated as median 50% embryo infective dose , was killed humanely. Some birds died between the intervals of clinical observation. From three selected culled or dead birds tissue samples  were collected into 15% w/v brain-heart-infusion broth (BHIB) with antibiotics  for RNA extraction for RT-PCR to examine virus dissemination in internal organs and for in-depth (deep amplicon) sequence analysis.Infection of turkeys has been described previously This has been described previously Each tissue sample collected in 1 ml BHIB was homogenised as a 10% w/v suspension using the QIAGEN Tissuelyser using 3 mm tungsten-carbide beads . Virus RNA was extracted from clarified tissue homogenate using the QIA quick RNA extraction kit (Qiagen) according to the suppliers’ instructions.5′-AGCAAAAGCAGG-3′). Each RT reaction was then amplified with PfuUltra™ II fusion HS DNA polymerase (Stratagene) and H7 HA specific primers (primer sequences are available on request), ligated into the pCR-Blunt vector (Invitrogen) and transformed into TOP10 E. coli (Invitrogen). Positive bacterial colonies were sequenced commercially  using the ABI 3730 XL (Applied Biosystems) Sanger sequencing platform.HA gene sequences were analysed using the deep amplicon sequencing approach described previously 5′-TCCGAGATATGTTAAGCAAGAGAGTCT-3′) and reverse (5′-AACCCGCTATAGCACC AAATAGG-3′) primers with two different probes. One ([FAM]-AACATTCTTCATCCCTGTTGCCAGCAG-[BHQ-1] was designed to detect both LPAI and HPAI virus sequences by hybridising to the HA coding region from nucleotides 970 to 996 of the H7 HA gene. The other was an “HPAI virus-specific” probe ([FAM]-CCTCTCCTCACACGCGA-[BHQ-1]) was designed to detect specifically HPAI virus sequences conserved in the HA of H7N1 HPAI viruses isolated from Italy during 1999–2000, and detects nucleotides 1015 to 1031 of these viruses  was employed in which two parallel PCR reactions on a cDNA template prepared from tissue and inoculum samples (described above) were set up using forward ( viruses . For bot5′-AGCAAAAGCAGG-3′) and the PCR step was performed using PfuUltra™ II fusion HS DNA polymerase (Agilent technologies) with a forward primer (5′-cgtatcgcctccctcgcgccatcag-NNNNNNNNNN-AAATGTCCGAGATATGTTAAGCAAGA-3′) consisting of a universal adapter sequence (primer A), indicated in lower case, a multiplex identifier sequence  and Italy/1279 HA specific sequence of 26 nucleotide bases (937–962). The reverse primer (5′-ctatgcgccttgccagcccgctcag-ATTTCCTGTTACTTGATCAATTG-CTG-3′) contained universal reverse primer B sequence (indicated in lower case) followed by Italy/1279 HA specific sequence of 27 bases (1171–1145). PCR amplification was performed in a T100™ thermal cycler (Bio-Rad) programmed for the initial denaturation step (95°C for 4 min), followed by 35 cycles of amplification  and a final extension (72°C for 10 min). PCR products were purified using gel electrophoresis and recovered using a gel extraction kit . After quantification with Quant-iT™ Picogreen® dsDNA assay kit (Invitrogen), all PCR products generated from each tissue sample were mixed together in equimolar concentrations and subjected to emulsion PCR using the GS FLX Titanium (Lib-A) MV emPCR kit (Roche Applied Science) according to the manufacturer’s instructions. Unidirectional sequencing of the amplicons from adapter A was carried out on a quandrant of a PicoTitrePlate on the Roche Genome Sequencer FLX (GS FLX) platform. The sequence data of amplicon reads was submitted to the European Nucleotide Archive (ENA).Ultra-deep sequence analysis of the HA gene cleavage site was performed using RNA samples extracted from Italy/1279 inoculum stocks and tissues  from infected turkeys. HA gene amplicons of 235 bp (bases 937–1171 of the HA open reading frame) were prepared by two-step RT-PCR described above. Briefly, the RT-step was performed using the Verso™ cDNA Kit (Thermo Scientific) with influenza virus universal oligonucleotide primer  and all birds had recovered from infection within 16 days pi. In contrast, in groups infected with 105.2 and 107.4 EID50 doses of the virus, all birds died or were culled for welfare reasons. In the 103.8 dose group, four (40%) birds died and the remaining six birds survived until the end of the experiment. In the groups that received 105.2 and 107.4 EID50 virus dose, all birds died or were culled as a result of influenza infection  that died on the eighth day following the first detection of virus from the same dose group (5.2 EID50 (Tissue samples were taken from three birds: a turkey (#83) that was directly infected and found dead on the fourth day after infection with 10.2 EID50 .in vivo tissues from severely affected birds was below 2.5% and perhaps considerably lower.We considered three possible reasons for death in turkeys infected with the LPAI H7N1 virus. First, the birds died of LPAI virus infection, second, there was evolution of a LPAI virus to a HPAI virus within the infected host, or third, there was heterogeneity of virus within the original virus inocula with a small population of HPAI virus in the LPAI virus stock. To address these options samples from designated tissues from the three selected infected birds were assessed by deep amplicon sequence analysis using standard Sanger sequencing of cDNA clones prepared from the amplicons. The sequences of these cDNA amplicons were compared to the sequences of the virus RNA in the inoculum and in buccal and cloacal swabs, as previously reported To explore further the possibility that HPAI virus might have been present at a lower level within the samples, we designed a dRT-PCR protocol for detection of HPAI virus in samples from LPAI virus infected birds. To set up the assay to detect HPAI virus contaminating sequences in the LPAI virus inoculum the assumption was made that either the virus could be contaminated with HPAI virus from the Italian outbreak or that if evolution occurred in the turkey host it could represent the sequences that emerged during the Italian outbreak of 1999–2000. The HPAI virus sequence-specific dRT-PCR was, therefore, based on the KGSRVRR*GLF sequence in HPAI virus isolates from the Italian outbreak . The LPAI and HPAI virus dRT-PCR assays was designed to be able to detect both LPAI and HPAI H7 HA sequences representative of viruses from the Italian outbreak using two Taqman probes analysed in parallel reactions. The amplicon for analysis spanned the HA1/HA2 cleavage site; one probe was designed to detect a conserved region in both LPAI and HPAI viruses in close proximity to the HA1/HA2 cleavage site; while the second probe was designed to detect, in the same amplicon, the additional 12 nucleotide insertion observed during evolution from LPAI to HPAI viruses in Italy in 1999–2000 outbreak .2 value) of 0.99. This reaction efficiency was calculated to detect reliably as few as 100 copies of the HA gene per reaction mixture (4.85 EID50) in HPAI-specific detection reaction samples did not interfere with the HPAI virus cDNA detection specificity or sensitivity, with the slopes and intercepts of the graphs being identical. The quantification showed that the system had a detection limit similar to the RNA levels present in 100.05 EID50 virus titres  virus and the equivalent region from the Italy/1279 (LPAI) virus. The amplification of 10-fold dilutions of HPAI and LPAI virus DNA detected by either the HPAI-specific probe or the H7 HA-common probe reproducibly displayed a linear relationship between Ct (cross threshold) values and HA copy number with a correlation coefficient  from three turkeys and from inoculum stock  were chaThe aim of the study described here was to investigate whether there was selective pressure that might result in the generation of HPAI viruses during replication of a LPAI virus within an infected host. The model based on infection of turkeys with a H7N1 LPAI virus from Italy collected in 1999 was chosen for several reasons. First, this outbreak has been studied in detail To examine the possibility that evolution of HPAI virus might have occurred in the infected host we investigated various explanations. The first possibility was that the virus stock used for the infection of turkeys could have contained a low titre of HPAI virus as a contaminating sub-species. We sequenced over 180 cDNA clones of the inoculum and found no evidence for the presence of a low level of HPAI virus and this was backed up by quantitative dRT-PCR and NGS in which no HPAI virus signature was detected in over 200,000 NGS reads.6 EID50 in the inoculum, and less than 100 DNA equivalent copies was detected in any tissue sample from the culled or dead birds by dRT-PCR analysis. The differential real-time RT-PCR would not detect viruses other than those for which we specifically designed the primers and TaqMan probes so analysis by NGS, using the Roche 454 sequencing platform, was used which allowed us to carry out deep-amplicon sequencing to a much greater depth and without any bias in the detection of sequences at the cleavage site. The NGS amplicons made from tissue samples contained a small number of reads (0 to 0.5%) with sequences containing a HPAI virus signature Click here for additional data file.Table S2Virus RNA titres in buccal and cloacal swabs collected from turkeys that died following Italy/1279 H7N1 virus infection whose tissues were selected for deep amplicon sequence analysis.(DOCX)Click here for additional data file.


The enormous genetic variability of human immunodeficiency virus type 1 (HIV-1) continues to present a major challenge for vaccine design and frustrate efforts to halt the epidemic. A proper understanding of this phenomenon is a prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines. In this study, we undertook a detailed molecular epidemiological investigation on HIV-1 vertically-infected children born from 1993 to 2008 in the state of São Paulo, Brazil.HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 48 participants. The near full-length genomic (NFLG) and partial fragments were determined by overlapping nested PCR and direct sequencing. The data were phylogenetically analyzed.Of the 48 samples  studied, 3 (6.2%) NFLGs and 39 (81.2%) partial fragments were successfully subtyped. Of the successfully subtyped sequences, 20 (47.6%) were subtype B sequences, 17 (40.4%) BF1 recombinants, and 5 (11.9%) subclade F1. Two of the partial BF1 chimeric isolates shared an identical recombination structure. Predictions of viral tropism using the computer program geno2pheno [co receptor] for phenotype prediction were determined for 15 subjects. X4 or X4 dual or mixed-tropic viruses were seen in 3 (20%) of participants and the V3 sequences of 12 patient virus strains (80%) were predicted to be R5-tropic virus.Our data provided evidence of unexpectedly high proportion of BF1 recombinants viruses transmitted from the first mother-to-child since the earliest days of the epidemic to the present time in Brazil. These findings offers additional insights to understanding the diversity of HIV-1 strains currently circulating in Brazil, with future implications for diagnosis, therapy, and efficient vaccine development.


To characterize parapoxviruses causing severe disease in wild ruminants in Stelvio Park, Italy, we sequenced and compared the DNA of several isolates. Results demonstrated that the red deer isolates are closely related to the parapox of red deer in New Zealand virus. Parapoxvirus  comprises several members: orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV), and parapox of red deer in New Zealand virus (PVNZ). PVNZ is responsible for a contagious pustular dermatitis in farmed red deer, with outbreaks reported only in New Zealand (Rangifer tarandus). Recently, the causative viruses of the clinical forms in reindeer were shown to be closely related to OV virus and PCPV, excluding the circulation of PVNZ in these countries  and ibex (Capra ibex) were closely related to OV, whereas the isolates from red deer (Cervus elaphus) grouped with PVNZ. Our findings provide new information about the diffusion of PPVs in wild ruminants and evidence that PVNZ is circulating outside New Zealand.To characterize the PPV agents causing severe disease in wild ruminants of Stelvio Park, we sequenced and compared the DNA of several isolates. Results showed that the viruses isolated from chamois  for identification of the causative agent of the disease. Viral particles, identified by morphologic features as PPV, were observed by negative stain electron microscopy in the material collected for pathologic examination from all the affected animals.B2L gene encoding a major envelop protein  gene of the red deer isolates . In the cases reported here, the disease was severe enough to cause the deaths of the animals. We cannot exclude that mild forms also can occur in wild red deer, and that only the most severe cases of the disease might have been brought to our attention; for this reason, additional data are needed to elucidate the clinical features of the disease in Italy. No information is available about the possibility of PVNZ natural transmission to domestic species, but inoculation of the virus into OV-naive sheep produced milder lesions than did OV (,Most PPVs are transmissible to humans, and these infections share clinical manifestations and exposure risks with other, potentially life-threatening zoonoses (


Sathuperi virus and is a possible ancestor of the reassortant Shamonda virus.Schmallenberg virus (SBV), an orthobunyavirus of the Simbu serogroup, recently emerged in Europe and has been suggested to be a Shamonda/Sathuperi virus reassortant. Results of full-genome and serologic investigations indicate that SBV belongs to the species  Genetic reassortment occurs naturally among these viruses, which results in the emergence of new virus strains that have altered biologic properties   as described . In addition to the newly determined sequences, published full-genome sequences of AKAV and Oropouche virus (OROV) from the National Center for Biotechnology Information reference genome database (www.ncbi.nlm.nih.gov/sites/genome) were used for sequence comparisons and the reconstruction of phylogenetic relationships. Coding sequences of each genome segment were aligned by using ClustalW  for codons, and phylogenetic analyses were performed by using maximum-likelihood methods in MEGA5  to 97.7%  . The L gSathuperi virus and is likely to be the ancestor of SHAV, which is in contrast a reassortant virus comprising the S and L segments from SBV and the M segment from another virus, as proposed previously . Neutralizing potencies of SBV antibodies against Simbu serogroup virus strains were tested by titrating virus in the presence of anti-SBV serum ; results were expressed as percentage titer reduction in relation to a parallel test without antiserum. Although DOUV and SATV were well neutralized, SHAV titers were not reduced at all , panel ASathuperi virus and that the species Shamonda virus should be renamed Peaton virus or Sango virus.Although our results do not support the suggestions of Yanase et al. (Sathuperi virus, our results show that the virus is most likely not a reassortant and is likely to be one of the ancestors of SHAV, whereas SHAV is a reassortant comprising the SBV S and L genomic segments and the M segment from an unclassified virus. These detailed insights into the phylogeny of SBV could be the basis for the development of efficient, cross-protective vaccines. Our results also highlight the importance of full-genome analyses to identify potential genetic reassortments and to investigate the evolutionary history of viruses with segmented genomes.In addition to showing that SBV belongs to the species


Zoonotic events of simian immunodeficiency virus (SIV) from non-human primates to humans have generated the acquired immunodeficiency syndrome (AIDS), one of the most devastating infectious disease of the last century with more than 30 million people dead and about 40.3 million people currently infected worldwide. Human immunodeficiency virus (HIV-1 and HIV-2), the two major viruses that cause AIDS in humans are retroviruses of the lentivirus genus. The genus includes arthritis-encephalitis virus (CAEV) and Maedi-Visna virus (MVV), and a heterogeneous group of viruses known as small ruminant lentiviruses (SRLVs), affecting goat and sheep. Lentivirus genome integrates into the host DNA, causing persistent infection associated with a remarkable diversity during viral replication. Direct evidence of mixed infections with these two closely related SRLVs was found in both sheep and goats. The evidence of a genetic continuum with caprine and ovine field isolates demonstrates the absence of an efficient species barrier preventing cross-species transmission. In dual-infected animals, persistent infections with both CAEV and MVV have been described, and viral chimeras have been detected. This not only complicates animal trade between countries but favors the risk that highly pathogenic variants may emerge as has already been observed in the past in Iceland and, more recently, in outbreaks with virulent strains in Spain. SRLVs affecting wildlife have already been identified, demonstrating the existence of emergent viruses adapted to new hosts. Viruses adapted to wildlife ruminants may acquire novel biopathological properties which may endanger not only the new host species but also domestic ruminants and humans. SRLVs infecting sheep and goats follow a genomic evolution similar to that observed in HIV or in other lentiviruses. Lentivirus genetic diversity and host factors leading to the establishment of naturally occurring virulent  Lentivirinae of the Retroviridae family. Retroviruses have been found in all classes of vertebrate animals, including fish, amphibians, birds and mammals [Lentiviruses are causative agents of many emerging and re-emerging infectious diseases threatening both animals and humans . These p mammals . In cont mammals . The len mammals . One of  mammals , or EquiSmall ruminant lentiviruses (SRLVs), comprising arthritis-encephalitis virus (CAEV) and Maedi-Visna virus (MVV) are a genetic continuum of lentiviral species that were initially isolated from goats and sheep, respectively . FollowiThe evidence of a genetic continuum of caprine and ovine field isolates from various geographical areas, that did not simply cluster according to the animal species from which they were isolated, demonstrates the emergence of new viral recombinants capable of overcoming species barrier and resulting in cross-species transmission/infection. Direct evidence of mixed infections under field conditions with these two closely related SRLVs was found, as suggested by detection of some subtypes in both sheep and goats . In natuMany emerging viruses partly escaping detection are already circulating in various hosts worldwide . This isSRLVs affecting wildlife have already been identified, demonstrating the existence of emergent viruses adapted to new hosts . These dversus avirulent infections, and emergence of new strains, pose a serious challenge to every aspect of SRLV control measures used to prevent the transmission of diseases between wild ungulates and livestock. Phylogenetic analysis and characterization of the biological properties of SRLVs adapted to wildlife , pol (polymerase) and env (envelope) genes. The SRLVs which encode auxiliary genes such as vpr, rev and vif are associated with the non-structural proteins with regulatory/accessory functions in viral replication [tat and shown to transactivate LTR promoters [vpr gene of HIV/SIV [Like others retroviruses, SRLVs are positive RNA viruses that copy their genomes into complementary DNA during their replication . The virlication . Unlike lication . The operomoters  was rece HIV/SIV  and lackpol gene encodes the enzymatic activities of reverse transcriptase (RT). RT is a RNA-dependent DNA polymerase that is essential for the conversion of viral RNA into DNA [The extent of genetic diversity depends on a set of factors, but variation arises ultimately through two major mechanisms: Mutation and recombination. The into DNA . Most reinto DNA .Recombination is a complementary mechanism of genetic variation which can assemble the beneficial genetic combinations more efficiently than mutation alone. Insertions and deletions in the lentivirus genome are also frequent occurrences due to high rates of virus replication . Togetheenv gene in an infected goat [On the other hand, dual infections (coinfections or superinfections) are an essential status for such recombination events ,9. Crossted goat . The infted goat . AlthougIn several countries, SRLVs strains have been analyzed and compared with current prototype strains of CAEV and MVV ,37,38,39in vitro cultivation properties revealed high virus replication efficiency in blood monocyte-derived macrophages but a slow/low virus replication in fibroblast-like cells. Virus replication/production could not be the explanation of their increased virulence [But outbreaks of disease with virulent strains kept re-emerging in susceptible populations in various parts of the globe. More recently, outbreaks with virulent strains associated with extensive neurological diseases were found in sheep of the central area of Spain ,41,42. Iirulence . The inhirulence ,44,45. Tin vitro following co-cultivation of infected macrophages with the non-permissive target cells [env gene, showed a different distribution of SRLV variants between blood and colostrum [Most transmissions of SRLVs among sheep or goats occur through the ingestion of virus infected colostrum ,44,45,46et cells . In goatolostrum . DiffereGenetic diversity also poses a relevant problem for SRLVs, testing and diagnosis ,49. The in situ hybridization in semen and various tissues of male genital tract suggesting a potential source of transmission [SRLVs have been detected by ELISA, PCR and smission ,53,54,55smission ,57. Molesmission ,22.The pSRLV’s mutagenic potential and diverse assortment of antigen epitopes have hampered vaccine development efforts . ImmunizSRLVs are widespread throughout the world causing serious economic implications for farmers and animal industry. SRLV infected animals produce less milk of poor quality, lose weight and are often prematurely culled . The resThere are millions of sheep and goats infected with SRLVs worldwide. The prevalence of SRLVs infections in North America and Europe ranges from 30%–80% compared to 0–10% in Africa and South America . ConsequBesides the high prevalence of infection in many countries, another major obstacle to eradication of SRLV is the ability of SRLVs to jump the species barrier. The first direct evidence came from natural interspecies transmission of SRLV subtype A4 between different animal species living under natural conditions . The facRecombination of different strains and subtypes contributes significantly to viral diversity and evolution both within individuals and populations ,27,67. SNumerous emerging infectious diseases, including zoonoses, were shown to originate from wildlife . DegradaIndependent zoonotic transmission events from NHPs to humans have generated two major virus types HIV-1 and HIV-2. HIV-1 remains a major global health problem with an estimated 33.3 million people living with the disease and over 30 million that have since died  due to ASmall ruminant lentiviruses (SRLVs) have a number of features in common with HIV-1 such as genome organization, long preclinical period following transmission of the virus, latency and persistence with a slow development of chronic degenerative disease, which may gradually lead to death . HoweverPrevalence of SRLVs often reaches 100% among infected flocks ,74,75,76Sheep, goats and cattle are major sources of high quality protein worldwide, and due to (I) rising incomes, (II) population growth, and (III) increased urbanization, there is a growing demand for meat and other animal products . Becausein vitro [In most countries crude milk is used as the raw substrate for dairy products . Since cin vitro . In an ein vitro .There is growing evidence of increased cross-species infection in mixed flocks worldwide resulting in the emergence of new variants adapted to new hosts and viral chimeras . DomestiAlthough incipient, there is still a possibility of persistence of SRLVs infection in human cells  that areCapra ibex) and mouflon (Ovis gmelinii) with SRLVs provide proof that these wild ruminants are susceptible for CAEV infection. Recovery of CAEV genetic sequences from Ibex that were in close proximity of grazing goat  a fatal naturally occurring CAEV infection was observed in four individuals. The CAEV was possibly transmitted to this species through ingestion of milk from infected domestic goats [Recent detection of cross-species infection and adaptation of domestic ruminant lentiviruses in free wild ruminants have further cemented this argument ,91. In Ric goats .Despite the proven efficiency of lactogenic transmission, the pastures are places where the transmission rate of infectious diseases between domestic and wildlife ruminants is the highest . The conDifferent factors can promote the constantly increasing interactions between wildlife and domestic animals. In Brazil, various endangered native goat breeds were infected with SRLVs through the introduction of exotic breeds to improve efficiency of milk production in local dairy herds ,93. Of nIn many countries, large populations of wild ungulates are concentrated in small delimited areas because of high human density and distribution . In addiIn order to successfully adapt to a new host, lentiviruses must evolve quickly to evade host defenses before they are transmitted to other individuals of the same species . HoweverIn domestic SRLV-free flocks, virus may re-emerge spontaneously. Serology tests in many cases also fail to detect antigenic variants by routine methods . LikewisWildlife and domestic animals are the major reservoirs for emerging and re-emerging pathogens, threatening both animals and humans . DegradaFurthermore, the emergence of infectious diseases within new hosts species or naïve populations can greatly influence species survival and adaptation. In fact, the cross-species infection of simian lentiviruses from non-human primates to humans has resulted in one of the most devastating infectious diseases (HIV/AIDS) of the last century. This demonstrates how a new viral pathogen can pose serious threats to public health . Recent Cross-species transmission between domestic small ruminants and wild species may affect the pathogenic and tropism properties of SRLVs, jeopardizing health and environmental safety. A continuous surveillance of wild small ruminant populations may improve our knowledge of the relationship between genetic evolution and new biological properties of emergent viruses, which in turn could generate biomedical advances and improved strategies for lentiviruses control in humans, domestic and wildlife small ruminants.Finally, the recent discovery that goats in Europe are readily infected with highly attenuated strains of CAEV that escape the detection tools and are not associated with disease development provides unique opportunity to use some of these strains as live-attenuated vaccines for goats to induce protection against the highly pathogenic strains. Since CAEV is not an infectious pathogenic virus in human, these highly naturally attenuated strains of CAEV could be used as a basis to derive live‑attenuated chimeric genomes as vaccines against the deadly HIV-1 in human.


In the past decade the concept of plant viruses as strictly disease-causing entities has been challenged. While the most well-studied and obvious interactions between plants and viruses are related to disease, there are several examples of mutualistic relationships between plants and viruses, both indirect and direct. These mutualistic interactions have not been fully explored, and many questions remain unanswered. One problem is the lack of knowledge of plant viruses in nature. Metagenomic surveys have estimated that only a small fraction of virus species are known. Additionally, globalization has led to the increased movement of plant material and virus movement. As viruses move from one area to another, new potential hosts offer the possibility of new interactions, both negative and positive.Curvularia thermal tolerance virus is a mycovirus that infects a plant fungal endophyte, Curvularia protuberata. When both virus and fungus are present in hot springs panic grass (Dichanthelium lanuginosum) the holobiont is able to grow in soil temperatures up to 65°C  Known-knowns: virus species or isolates that are already known to be in the environment being surveyed; (2) Unknown-knowns: new virus species or isolates of a known family, or known viruses that have not been found previously in the surveyed environment and; (3) Unknown-unknowns: viruses that are completely novel and share little to no sequence similarity with other known viruses. Sequencing data for each instance can be analyzed differently based on the questions being addressed. The removal of non-viral sequences from the sample either before or after sequencing will, of course, increase the chances of identifying viruses within a metagenomic sequence dataset, so care should be taken in both sample preparation before sequencing and manipulation of sequence data after sequencing. Methods to enrich for plant viral-specific sequences include isolation of virus-like particles (Muthukumar et al., The large amount of sequencing data that shares little or no nucleotide similarity with known sequences in curated databases such as GenBank suggests that there are still many unknown microbes that have yet to be described, with unknown-unknown viruses likely to be prevalent among them. These unknown sequences continue to be difficult to identify and will require new and novel methods to assign to a taxon. There have been significant efforts to describe these unknown-unknowns in different environments, with new analysis methods tailored for virus discovery either by sequence similarity or by clustering genes (Williamson et al., de novo assembly is essential as genome mapping or BLAST assisted assembly is not an option. Assembly programs tailored for virus de novo assembly have been created, and modifications to assembly processes have been used to generate fully assembled genomes of extremely low titer microbes (Albertsen et al., Unknown-unknown viruses share little or no sequence similarity with known viruses, so quality There is still much to be discovered on the topic of plant-microbe interactions, and of plant-virus interactions in particular. Metagenomics offers us a unique tool to elucidate the current state of viruses in plants and the role viruses play in these interactions. When these studies are done on individual plant samples rather than pooled samples from a larger environment, a system known as “ecogenomics” (Roossinck et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many viruses use multiple receptors: some of which are unique to a certain cell type, whereas others are found in many cell types. After the virus enters into cells, various cellular proteins may interact with it; some support virus replication, while others inhibit it. Once virus succeeds to establish its life cycle in the target cell, the progeny viruses disseminate within the tissues or systemically via viremia. The intrinsic pro- or anti-viral cellular machinery differs among cell types. Thus, depending on the receptors used, the viral cell tropism is determined, resulting in the characteristic distribution of virus-infected cells/tissues and the disease outcome in vivo.How viral cell tropism, determined by the receptor used, can affect the disease outcome in acute and chronic virus infection is a major subject under extensive investigation in Virology. To understand the mechanism which causes human diseases by viruses, that is, viral pathogenesis, we have studied various aspects of virus infection at a cell/tissue level or using animal models. In this context, the recent development of reverse genetics allows us to visualize virus-infected cells/tissues or even the virus itself. By applying such manipulated viruses to animal models, it is also possible to analyze the dynamics of virus infection In this Research Topic, we selected on studies connecting virus receptor usage and the pathogenesis of various viruses causing acute or chronic infection. Eventually, it could expand to cover the receptor-pathogenesis relationship in various acute and chronic virus infection. This research topic comprises an original research article on HIV-1, two opinions articles on the hepatitis C virus (HCV) and norovirus, while the remaining review articles on HTVL-1, measles virus (MV), mouse hepatitis virus (MHV), influenza virus, HCV, and enterovirus (EV) provide overviews on various aspects of viral pathogenesis. In all these review/opinion articles, at least one comprehensive table or figure is incorporated so that readers who are unfamiliar with these viruses can get a message at a glance.+ T cells with distinct differentiation phenotype including CCR5+ memory. In contrast, a receptor for HTLV-1 and related pathogenesis is still intriguing issue, which is described by Hoshino (With regards to the cell tropism of HIV-1, Terahara et al.  presente Hoshino  in his ein vivo which may influences the disease outcome using monkey models, while Takeda et al. (The two reviews on the MV (Kato et al., a et al.  discusseIto et al.  addresseNishimura and Shimizu  and YamaAs regards to the virus recognizing sugar moieties, Ramos and Fernandez-Sesma  providedWe will learn by these articles the fact that to identify a receptor is the first important step to know a virus, but many questions remain in order to fully understand human diseases caused by viruses. I would like to express my cordial thanks to all the contributors for this topic. I hope readers find the content interesting, but most importantly, that the information will prove very useful for future research.


CASP1 variants suffer from systemic sterile inflammation despite reduced secretion of IL-1ß. As previously demonstrated by our group CASP1 variants lead to reduced enzymatic activity of procaspase-1 by destabilizing the tertiary structure of the caspase-1 tetramer. A possible explanation for an alternative pro-inflammatory pathway has been provided by Lamkanfi and colleagues indicating an association between enzymatically inactive procaspase-1 and receptor interacting protein kinase 2 (RIP2) leading to NF-kB activation.Patients with unexplained recurrent febrile episodes and CASP1 variants interfere with the IL-1β production or release and lead to the activation of alternative pro-inflammatory pathways.The objective of this project is the identification of possible subcellular mechanisms how Using confocal microscopy, in vivo live cell imaging and an in situ proximity ligation assay we analyzed the subcellular distribution of procaspase-1 wildtype and mutants as well as the interaction with RIP2 in naïve or virally transduced THP-1 cells.CASP1-L265S. Procaspase-1 activation, initiated by the assembly of multiprotein complexes (inflammasomes), was induced by stimulation with LPS and Nigericin. First results suggest disturbed microvesicle shedding from CASP1-L265S expressing cells after administration of Nigericin. In addition to live cell imaging, the interaction of procaspase-1 and RIP2 has been studied in vitro in naïve THP-1 cells using antibody labeling and proximity ligation assay showing a time dependency after LPS stimulation.THP-1 cells were virally transduced with GFP- or mCherry fusion proteins of procaspase-1 wildtype and variant This result suggests a possible influence of procaspase-1 variants on plasma membrane properties, pyroptosis and the release of microvesicles.None declared


Following careful evaluation of the relevant dataset, each of the points brought up by Young et al. has been addressed in this response. We anticipate this will clarify our findings regarding ERVmch8, an ecotropic endogenous retrovirus that was shown to have cerebellum-specific and age-dependent expression patterns in C57BL/6J mice.This correspondence was written in response to the comments by Young  At that time, the annotated Emv2 locus did not contain any sequences similar to mouse endogenous retroviruses. Interestingly, a survey of the latest NCBI database (Build 37.2) for the Emv2 locus on December 08, 2011, in response to the correspondence by Young et al., yielded no annotated information about the Emv2 locus, and it appears that the previous annotations may have been removed. During the course of our analysis regarding the Emv2 locus in the C57BL/6J mouse genome, screenshots of the data were captured and recorded, and revealed the annotation of the Emv2 locus on chromosome 8, which is distantly located from the ERVmch8 locus .As stated in the paper by Lee et al.:This was cited in the paper by Lee mch8 locus (~78.4 cM), according to a survey of the NCBI map viewer ... mch8 shares the same locus on chromosome 8-qE1 region with Emv2 with an assumption that the NCBI annotation information regarding the Emv2 locus needs to be revisedUnexpectedly, we were unable to retrieve the nucleotide sequence, which is presumed to be the Emv2 provirus, from the Emv2 locus annotated in the ... However, it is still a possibility that ERV.et al., we were not able to find the "full/complete" sequence information from the NCBI database using the keyword "Emv2", and it was unclear why there was no "full/complete" sequence information deposited into the database when substantial work in regard to Emv2 has been reported. When preparing the manuscript, a thorough survey of relevant scientific publications for Emv2 also yielded neither "full/complete" nor partial sequence information.As indicated in the paper by Lee mch8 locus indeed matches the Emv2 locus. Importantly, the difference in distance between the annotated Emv2 locus (chromosome 8 at ~67 cM region) and the ERVmch8 locus (chromosome 8 at ~78.4 cM region) needs to be clarified  in the title of Figure mch8 locus  retrieved only one locus for ERVmch8, which is located on chromosome 8 of the C57BL/6J mouse genome and the only copy of an ecotropic MuLV found in that genome. The data presented in the paper by Lee et al. intend to only show the general distribution of the 'ERVmch8 sequence' throughout the genomes of 57 mouse strains and do not specifically refer to an ERVmch8 sequence on chromosome 8 (since the copy number or locations can vary depending on the strain). As stated in the results section of the paper by Lee et al., the variability of size and intensity of these bands suggests "polymorphisms in the sequences and/or copy numbers."The primer set used in the paper by Lee It should also be noted that both the Southern blot and PCR protocols share a specific step which is "nucleic acid" to "nucleic acid" hybridization. As such, both protocols harbor the potential for false identification of the intended target DNAs. Our laboratory also routinely includes relevant RT-negative controls for RT-PCR analyses.et al. clarify the comments brought up by Young et al. We anticipate this correspondence will clarify our findings regarding ERVmch8, an ecotropic endogenous retrovirus that was shown to have cerebellum-specific and age-dependent expression patterns in C57BL/6J mice.We hope that the responses by Lee The authors declare that they have no competing interests.The authors contributed equally to this article.


Additionally, we discuss the variation in vivo of selected isolates of Old World arenaviruses, particularly after infection of different animal species. We also discuss the recent emergence of new arenaviruses in the context of our observations of sequence variations that appear to be host-specific.Arenavirus particles are enveloped and contain two single-strand RNA genomic segments with ambisense coding. Genetic plasticity of the arenaviruses comes from transcription errors, segment reassortment, and permissive genomic packaging, and results in their remarkable ability, as a group, to infect a wide variety of hosts. In this review, we discuss some  Arenaviridae, the Filoviridae and the Bunyaviridae share some structural motifs that could be derived from common ancestry or functions. Classical studies on the prototypic arenavirus species, lymphocytic choriomeningitis virus (LCMV), have followed the sequence and phenotypic variation of the LCMV-Clone 13 and LCMV-Armstrong strains in vivo and in vitro. Such studies shed light on our own analyses of the Mopeia/Lassa reassortant virus, ML29 as it varies after passage through a variety of host species.Here we review genetic and phenotypic variations of arenaviruses at the level of virus families, at the level of genera, at the level of species, and finally within those entities known as “strains” or “isolates”. Sequence analysis reveals that the Arenaviruses have enveloped particles containing bi-segmented genomes of single-stranded RNA encoding four viral proteins in an ambisense manner. The small segment called S ~3.4kb), encodes the glycoprotein precursor (GPC) and the nucleocapsid protein (NP) that are the most important immunogens of the virus . The NP .4kb, enctrans-acting virus factors required for replication and transcription [cis-acting elements for RNA replication and transcription [The Large (L) segment ~7.2Kb), encodes the small zinc-binding protein (Z) that functions as a matrix protein, interacts with L and NP and other host proteins, plays a role in viral transcription and replication, has pro-apoptotic activity and is essential for virus budding ,12,13,14Kb, encodcription ,16. L ancription . Both RNcription ,17,18  established the family rameters ,22. Thisrameters . The aredontinae . It is hdontinae .Arenaviridae into NW and OW groups, species demarcations are determined by the following criteria of their member viruses: significant differences in antigenic cross-reactivity and cross-neutralization; role as an etiological agent of disease (or not) in humans; presence in a defined geographical area, host species or group of species, and significant protein sequence differences compared to other viruses in the genus . However, there are still some poorly-defined criteria for classification. For example, consider the criterion that the amino acid sequence of the NP has less than 88% homology to the closest arenavirus. It is interesting that some Lassa isolates vary more than 12% from each other, and should be classified in different species but they are not. Another critical point is the possibility of reassortment among viruses assigned to the same species. It is odd that Lassa and Mopeia were able to reassort, even though they are assigned to different species, perhaps due to compatibility between the Lassa and Mopeia terminal sequences. In addition, there are several reports of arenaviruses detected by molecular techniques from human or animal samples, however some of them are not yet associated with human diseases or failed to replicate in cell cultures  females with MACV resulting in female sterility [Neotoma albigula) pregnant females were seronegative for WWAV in a population with 60% seropositivity, indicating that little vertical transmission occurred. Similar observations are reported for the drylands vesper mouse -Junin virus disease model, where transmission is more common between adult males and associated with the presence of scars, implying violent transmission during aggressive encounters [Mastomys sp. with LASV seems to have no sex or age bias suggesting more frequent vertical transmission from dam to pups [Arenaviruses normally produce persistent infections in rodents, with chronic viremia and viruria, spreading virus through urine, feces, and saliva to other rodents and accidentally infecting humans or other mammals ,42,43. Aterility . In othecounters ,49,50. I to pups . From thMuridae, or hamsters from the family Cricetidae. African viruses circulate in endemic areas between rodents from the family Muridae, genus Mastomys, Praomys, or Arvicanthis. The American arenaviruses circulate in specific areas between rodents from the family Cricetidae, genera Oryzomys, Sigmodon, Neotoma, Nephelomys, Oecomys, Calomys, Zygodontomys, Neacomys, or Akodon (Artibeus sp.)  . The facaviruses . In the aviruses . Similaraviruses ,56. Arenavirus-rodent interactions exemplify virus-host codivergence, in which one rodent species diverges from another so much, that the viruses of each host are no longer able to infect the same hosts equally well. Codivergence and cospeciation are similar: if two species are closely associated, as in viruses and their hosts, they might speciate or evolve in parallel. Thus it could eventually be possible to assign host species by the population of parasites (including viruses) they carry . An alteTen arenaviruses are known to cause natural  or laboratory acquired  human disease in specific geographical regions . Those vThere is also evidence of selective pressures on human populations by arenaviruses. Two genes related to LASV infectivity and immunity (LARGE and interleukin 21) had been associated with a possible positive selection after viral infection leading to LF resistance. Those genetic traits are evidence that the human incidental host co-evolved with LASV. In addition, LASV infection in pregnant women results in nearly 100% fetal lethality exerting a strong selective pressure in the endemic regions. Thus, arenaviruses exert selective pressures on both human and carrier host populations ,61.vs. transferrin receptor 1), different entry mechanism (delivery to endosomes via vesicular trafficking vs. endocytosis), and different protective immune responses in rodent and in primate incidental hosts (innate vs. acquired immunity) [During the codivergence process, OW and NW arenaviruses evolved to use different cell receptors  ,66,67,68in vitro, those experiments indicated that there are restrictions that prevent recovery of all possible combinations [-3 to 10-5 per nucleotide incorporated during replication [in vitro estimate that the error frequencies of the LCMV RdRp are between 1 × 10-4 and 5.7 × 10-4 substitutions per nucleotide [The arenaviruses, like other RNA viruses, are highly divergent due to high mutation rates from a low-fidelity viral RdRp and due to reassortment and possibly recombination events that contributed to viral diversification during arenavirus evolution ,69,70. Ainations ,74,75,76inations ,70,77,78lication ,80. Lethcleotide ,82. Thoscleotide . Comparicleotide , even ifcleotide ,86; and cleotide ,86. Sequcleotide . LASV iscleotide ,88. The in vitro changes should occur at a different rate than those seen in vivo, due to the absence of several selective forces .When referring to a virus strain we are describing the most abundant variant from a closely-related virus swarm containing individual particles with broadly-distributed mutations. A viral isolate containing many variants, called quasi-species, could act as a unit of selection through a continuous dynamic process of genetic variation, competition, and selection . HoweverSeveral studies explore arenavirus variability under specific selective pressures as a model of viral evolution. For instance, serial passages of LCMV in the presence of a mutagen such as 5-fluorouracil (5-FU), and an anti-viral like ribavirin leads the viral population into extinction in an event known as “error catastrophe” or “lethal mutagenesis” ,99,100. in vitro passage, though none of the SNPs exceeded 25% frequency within the viral population [Cell culture manipulations of Lassa and Mopeia viruses revealed that these African viruses species were so closely related as to be able to reassort with one another . Initialpulation . This agpulation .in vitro, increasing or decreasing mutation rates, can affect virus fitness and viral tropism and produce a spectrum of outcomes depending on particular conditions of the virus-host interaction. Those findings were explored with the goal of using “lethal mutagenesis” as an anti-viral treatment, and viral clones with high fidelity polymerases as attenuated vaccines [Thus, changes in virus diversity vaccines ,108.in vivo [Chronic infections are the best models for studying the structure and evolution of a virus population within a single host since viruses have a chance to evolve after extensive replication, genomic changes and selection. The prototypic virus LCMV is able to produce acute or persistent infections in mice and has been used as a model to study viral population dynamics in vivo ,110. Thein vivo ,119,120.7 PFU/ml [-4 substitutions per nucleotide and a virus of approximately 104 nucleotides in size, by the time maximum viremia is reached  every possible mutation had multiple chances to occur. In fact there is a close correlation between arenavirus concentration and severity of disease. Analysis of cytotoxic T lymphocyte or neutralizing antibody escape mutants in mice, showed mutation rates around 3 × 10-4 substitutions per nucleotide and suggested that those mutations are present and are selected from the virus stock instead of appearing spontaneously [During the peak of an arenavirus acute infection in monkeys, the number of viral particles in blood is around 1 × 107 PFU/ml ,122,123.aneously ,125,126.aneously ,127,128.Sequence comparison of viral isolates from those two tissues (brain or lymphoid/spleen) revealed five nucleotide changes but only two amino acid differences: one at residue 260 of GP1, phenylalanine (F) to leucine (L); and the other at residue 1079, lysine (K) to glutamine (Q) in the L protein ,130,131 Table 1A) Nucleotide and protein single nucleotide polymorphisms (SNPs) from the S segment. (B) Nucleotide and protein SNPs from the L segment. Black color letters represent the consensus sequence. Green, blue, and red letters represent abundance of that mutation at nearly 20%, 40% and 100%, respectively. Blue dark squares are the reported differences between both virus strains as follows: In the S segment nucleotide mutation 603 corresponds to amino acid 177, nucleotide mutation 855 corresponds to amino acid 260, nucleotide mutation 1298 corresponds to amino acid 407; in the L segment nucleotide mutation 3797 corresponds to amino acid 1079, nucleotide mutation 1798 corresponds to amino acid 412. The light blue square holds the previously reported mutation and confirmation of its presence in our laboratory strain. The Salvato 2012 pyro-sequencing shows diversity in those viral clones even within the same clone from the same laboratory.LCMV sequence diversity. Comparison between different isolates of LCMV-Armstrong and LCMV-Clone 13.  in persistently-infected C3H/st mice. A close analysis of plaque-purified viral clones illustrated that the virus strain contained variants of different phenotype. Less than 5% of the viral isolates were able to replicate to high titer in GH-producing cells and cause GHDS in C3H/st mice. The GHDS phenotype was due to a G to A nucleotide substitution at position 535  of the LCMV-WE GP. However, the stable presence of GHDS-phenotypically silent variants in the LCMV-WE viral inoculum was not enough to induce disease, indicating that pathogenic variants can be maintained within a non-pathogenic viral population. Experimental mixtures of LCMV WEc54 (serine) and WEc2.5  showed that the proportion of the latter has to be between 10% and 50% in order to induce GHDS. In addition, this study found variability even inside each cloned virus ,94.in vivo. After inoculation of adult mice, all animals showed the original pathogenic pattern. Viruses from lymphoid organs (Clone13-like) caused persistent infections and immunosuppression, in contrast to virus isolated from CNS. Those findings suggested that the immune system is not the main selective force and sequence analysis showed again that the mutation in the position 260 was tissue specific (F260L or F260I associated with lymphoid organs). Additionally, LCMV-Clone13-like viruses were found only in cells in the marginal zone and white pulp of the spleen, specifically in CD11c (+) and DEC-205 (+) splenic dendritic cells; while LCMV-Armstrong-like were found in the red pulp in CD11c (-) and DEC-205 (-) splenic dendritic cells. The specific tropism of those mutants was associated with increased receptor GP1/α–DG affinity. LCMV- Clone 13 (F260L or F260I) and LCMV WEc54 (S153) showed around 2–3 logs higher affinity for α–DG than their parental virus LCMV ARM and LCMV WEc2.2 (S153F) [in vivo, defining the disease outcome. Additional selective pressures include enzyme processing rates, different nucleotide concentrations, codon bias, dose and route of infection and other microbes in the host. For example, there is some contribution from the arenavirus polymerase to the induction of persistence by LCMV-Clone 13, in that its mutation K1079Q gives it preferential ability to replicate in plasmacytoid dendritic cells over LCMV-Armstrong [In order to evaluate the role of the immune system in selecting variants of LCMV, different isolates from tissues of immunocompetent and immunocompromised animals were tested  (S153F) ,136. Thormstrong .In conclusion, the analysis of specific mutations focuses on the function of each viral protein or genome component and its biological significance. However, the analysis of complex viral populations is in its infancy, and it could give a broader picture of how mixtures of particles, or quasi-species, impact pathogenesis and immunity. Therefore, the use of new tools such as deep sequencing and mathematical models can approach the pharmacological and vaccine research from a more holistic angle.In addition to their natural hosts, arenaviruses can infect other animals including hamsters, rabbits, squirrels, guinea pigs, dogs, chickens, bats, and primates. Some of them are refractory to disease ,143,144.in vivo, rhesus monkeys, marmosets, and mice were vaccinated and followed for several weeks by isolation of serum virus. Isolates were characterized by pyro-sequencing and showed several SNPs indicating the heterogeneity within cloned viral populations. As mentioned previously, the variation after 12 passages in vitro was less than 20% and within the expected mutation rates of arenaviruses. Some animals developed a very low viremia during the first two weeks after vaccination. ML29 virus isolated during these first few weeks was subjected to pyro-sequencing and proved to have a lower number of SNPs (less variation) than virus obtained from in vitro passage, and surprisingly, some of the variations were host-specific , and we took the opportunity to examine sequence variation with the goal of finding host-specific variants. In order to study the complexity and stability of ML29 specific . The virspecific .Viral infection starts with binding to cellular receptors on the host cell. The virus:receptor interaction plays a role in tropism, host-range and pathogenesis. As discussed above, one amino acid change in the arenaviral GP or RdRp gives the virus the capacity to infect different cells and alters viral pathogenicity ,130,147.Sequence analysis of recently emerging viruses suggested that genes from arenaviruses share some homology with other negative-strand virus such as filoviruses and bunyaviruses ,154. TheA new type of bi-segmented negative-strand RNA virus has recently been discovered in snakes. Three isolates of the new virus are characterized by ambisense coding and some sequence-homology with arenaviruses. The snake virus L and NP genes are homologous to those of arenaviruses, the GP sequences are homologous to filovirus envelope glycoproteins, and the Z gene sequences are homologous to host ubiquitin ligase . Since tAnother possible source of arenavirus variation comes from co-infection with retroviruses or infection in the presence of a reverse transcriptase/retrotransposase. This could result in the integration of arenavirus sequences into the somatic genomic material of a host organism. The fact that this phenomenon takes place has resulted in speculation about its effect on low-level arenaviral gene expression leading to self-tolerance of arenaviral antigens ,161.Outside the laboratory, the real world is a very complicated microbiome, and the few examples of co-mingling of arenavirus sequences and host or other microbe sequences broadens the possibilities for arenavirus evolution.In conclusion, viral genetic diversity plays an important role in the viral population fitness that is shaped by different selective pressures in the host, determining the host range of the viral offspring and the outcome of the infection. Therefore, it is important to study the heterogeneity and complexity of viral populations in the context of viral evolution and pathogenicity. Such observations will constitute a model for developing better viral classification systems, better vaccine candidates or vaccine screening techniques, and new drugs for viral treatments.


Cryptococcus gattii, which is causing an outbreak in the Pacific Northwest region of North America, causes life-threatening pulmonary infections and meningoencephalitis in healthy individuals, unlike Cryptococcus neoformans, which commonly infects immunocompromised patients. In addition to a greater predilection for C. gattii to infect healthy hosts, the C. gattii genome sequence project revealed extensive chromosomal rearrangements compared with C. neoformans, showing genomic differences between the two Cryptococcus species. We investigated the roles of C. gattii calcineurin in three molecular types: VGIIa (R265), VGIIb (R272), and VGI (WM276). We found that calcineurin exhibits a differential requirement for growth on solid medium at 37°, as calcineurin mutants generated from R265 were more thermotolerant than mutants from R272 and WM276. We demonstrated that tolerance to calcineurin inhibitors  at 37° is linked with the VGIIa molecular type. The calcineurin mutants from the R272 background showed the most extensive growth and morphological defects (multivesicle and larger ring-like cells), as well as increased fluconazole susceptibility. Our cellular architecture examination showed that C. gattii and C. neoformans calcineurin mutants exhibit plasma membrane disruptions. Calcineurin in the C. gattii VGII molecular type plays a greater role in controlling cation homeostasis compared with that in C. gattii VGI and C. neoformans H99. Importantly, we demonstrate that C. gattii calcineurin is essential for virulence in a murine inhalation model, supporting C. gattii calcineurin as an attractive antifungal drug target.The pathogenic yeast  Cryptococcus gattii and Cryptococcus neoformans are closely related basidiomycetous yeast species that cause the disease cryptococcosis after inhalation by the host. Cryptococcosis can cause pneumonia and disseminates throughout the body to infect other tissues, especially the central nervous system, causing meningoencephalitis  patients, C. gattii frequently causes disease in both immunocompetent and immunosuppressed hosts  in C. gattii   (A. fumigatus and C. dubliniensis) (Magnaporthe oryzae)  and regulatory B calcium-binding subunit (Cnb1). Active calcineurin is an AB heterodimer, and loss of the B subunit can result in destabilization of the A subunit, causing calcineurin malfunction (Guizotia abyssinica) agar plates was made with 70 g of ground niger seed in 350 mL of distilled water (dH2O), filtered through cheese cloth with the addition of 1 g of dextrose, 20 g of Bacto agar, and dH2O added to 1 L. L-DOPA agar plates was made with 20 g of Bacto agar autoclaved in 900 mL of dH2O followed by addition of 1 g of L-asparagine, 1 g of dextrose, 3 g of KH2PO4, 0.25 g of MgSO4, 100 mg of L-DOPA, and 1 mg of thiamine. The pH was adjusted to 5.6, and dH2O was added to 1 L. Low iron media was made with 5 g of dextrose, 5 g of asparagine, 400 mg of K2HPO4, 80 mg of MgSO4, 250 mg of CaCl2, and 20 mg of ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) followed by the adjustment of pH to 7.4 in 1 L of dH2O; then, 1 mL of 1000× vitamin-mineral mixture  was added after autoclaving.Yeast strains used in this study are listed in C. gattii orthologs of the gene encoding the C. neoformans calcineurin catalytic subunit (Cna1) were identified by reciprocal BLAST searches between the two species, with the reciprocal best BLAST hit orthologs in C. gattii being CNA1 . C. gattii R265 Cna1 shares 99.4% and 97.0% identity over the full protein length with its corresponding C. gattii WM276 and C. neoformans H99 orthologs, respectively . In addition to the catalytic domain, C. gattii Cna1 has the conserved calcineurin B binding, calmodulin binding, and autoinhibitory domains .The  domains  , R272 (VGIIb), and WM276 (VGI) strains. The nourseothricin (NAT) resistance gene from plasmid pAI3 was used as a dominant selectable marker in a biolistic transformation protocol with a Bio-Rad model PDS-1000/He biolistic particle delivery system (Table S1. The cna1Δ mutant was generated in the C. gattii strains R265 (VGIIa), R272 (VGIIb), and WM276 (VGI) by overlap PCR as previously described  was amplified with the M13 universal primers (JC65/JC66) from plasmid pAI3. The CNA1 gene replacement cassettes were generated by overlap PCR with flanking primers JC178/JC179, precipitated onto gold microcarrier beads , and the VGII strains R265 and R272 were biolistically transformed.All deletion strains were generated from the prototrophic CNA1 gene were amplified with primers JC207/JC208 and JC209/JC210 from genomic DNA. The CNA1 replacement cassette was generated by overlap PCR with primers JC211/JC212, and WM276 was biolistically transformed. Stable transformants were selected on YPD medium containing 100 µg/mL nourseothricin. Two independent nourseothricin-resistant cna1 mutants  derived from two separate transformations were obtained , CNA1 gene, and 3′NCR. Because calcineurin is essential for growth at 37°, we screened transformants in YPD medium at a slightly more stringent condition of 38°. Transformants able to grow at 38° were colony purified and replica-plated to YPD medium containing 100 μg/mL nourseothricin. Both nourseothricin-sensitive and -resistant colonies were observed. The nouseothricin-sensitive colony YC875  represents native locus complementation from which the NAT selectable marker within the cna1 mutant YL165 was replaced by wild-type CNA1 gene (thus resulting in nourseothricin sensitivity), whereas nouseothricin resistant colonies represent ectopic CNA1 integration , or suppressor mutations .4 in the same buffer for 2 hr on ice in the dark. Cells were again rinsed with three changes of buffer as described previously. After the last buffer wash, the cells were centrifuged and the pellet was embedded in warm (60°) 2% agarose prepared in the same buffer. The tubes were spun for 5 min while the agarose was warm enough to pellet the cells before the agarose gelled. After 1 hr on ice, the gelled pellets were removed from the tubes and cut into 1-mm3 blocks in dH2O at room temperature. Blocks were placed in buffer at room temperature and then rinsed once with room temperature dH2O before en bloc staining with 1% uranyl acetate in dH2O in the dark for 1 hr at room temperature. Samples were rinsed once with room temperature dH2O and then dehydrated through a graded ethanol series  for 1 hr each change and then infiltrated with Spurr’s resin  for at least 6 hr each. Individual pieces were embedded in fresh 100% Spurr’s in BEEM capsules at 70° for 2 d. Thin sections were cut with an LKB NOVA Ultrotome III , collected on 200-mesh grids, and then stained with 4% aqueous uranyl acetate for 1 hr and Reynold’s lead citrate for 4 min. Grids were viewed using a JEOL 1200EX transmission electron microscope . Images were recorded on Kodak 4489 film  that was then scanned using an Epson 4870  flatbed scanner at 1200 dpi. Digital negatives were processed to positives and labeled using Photoshop CS4.Cells were pelleted and rinsed in PBS buffer and resuspended in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH = 6.8) at room temperature for 1 hr. The cells were then transferred to 4° and incubated in fixative for several weeks. The cells were rinsed with three changes of cold 0.1 M sodium cacodylate buffer (pH = 6.8) by centrifugation and resuspended in fresh buffer and then postfixed with 2% OsOC. gattii melanin production. Strains were grown overnight at 24°, washed twice with dH2O, diluted to 1 OD600/mL, and then 5 µL of cell suspension was spotted on Niger seed and L-DOPA agar plates and incubated for 72 hr at 24°.Niger seed and L-DOPA plates were used to determine 2O, and diluted to 0.2 OD600/ml (5 mL) in low iron media for growth at 24° for 72 hr. Three microliters of India ink  was added to 97 µL of cell suspension. The images were taken at 1000× magnification.Strains were cultured in YPD media overnight at 24°, washed twice with dHAnimals studies conducted in the Division of Laboratory Animal Resources facilities at Duke University Medical Center (DUMC) were conducted in good practice as defined by the United States Animal Welfare Act and in full compliance with the guidelines of the DUMC Institutional Animal Care and Use Committee. The vertebrate experiments were reviewed and approved by the DUMC Institutional Animal Care and Use Committee under protocol number A217-11-08.6 cells per milliliter. Dilutions of the cells were plated onto YPD and incubated at 24° for 48 hr to determine CFU and viability. Groups of 10 mice  per strain were anesthetized with pentobarbital , and inoculated with C. gattii via intranasal instillation of an inocula of 5 × 104 cells (in 50 µL). Survival was monitored 1 to 2 times daily, and moribund mice were killed with CO2. Kaplan-Meier survival curves were generated with Prism 5.03 , and P values were evaluated by a Log-rank (Mantel-Cox) test. A P value of <0.05 was considered significant.Seven- to eight-week-old female A/Jcr mice  were used in this study. For infection, strains were cultured in YPD broth overnight at 24° and washed twice with sterile phosphate-buffered saline (PBS). Cells were counted with a hemocytometer and resuspended in sterile PBS at 10C. gattii infected mice (n = 5 for each strain) were dissected at day 14 after infection. Half organ portions were weighed, transferred to a 15-mL Falcon tube filled with 5 ml PBS, and homogenized for 10 sec at 13,600 rpm/min . Tissue homogenates were serially diluted, and 100 µL was plated onto a YPD plate containing 100 µg/mL chloramphenicol. The plates were incubated at 24° for 48 to 72 hr to determine CFU per gram of lung or brain. The significance of differences in fungal burden was determined using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test. For histopathological analysis, half organ samples of lung and brain were fixed in 10% phosphate-buffered formalin (Fisher Scientific), and mucicarmine and hematoxylin & eosin (H&E) stainings were performed by technicians at the Department of Pathology at Duke University. After slide preparation, each sample was examined thoroughly by microscopy for analysis of Cryptococcus colonization (mucicarmine) and tissue necrosis (H&E). Images were captured using an Olympus Vanox microscope .To determine fungal burden, the lungs and brains of Galleria mellonella) of the final instar larval stage (~0.3 g) from Vanderhorst Wholesale, Inc.  were used (10 per strain) within 7 d from the day of shipment. The larval infection protocol was adapted from previously described methods for C. neoformans  on C. gattii growth at room (24°) and body (37°) temperatures. We found that C. gattii R265, R272, WM276, and C. neoformans H99 wild-type strains exhibited different responses at 37° in the presence of FK506 or CsA  FK506 is either not efficiently imported into the cells, or is being rapidly exported; or (3) FKBP12 is not sufficiently abundant to inhibit all calcineurin activity.The ability to grow at host body temperature (37°) is a critical virulence factor for pathogens to establish infection. Previous studies on Sod1, Tps1, Tps2, Pka1, Pka2, Ste12α, and Gat1 have revealed examples of divergent functions between 6 or CsA . C. gatt strains . This fiC. gattii isolates representing the VGIIa (R265), VGIIb (R272), and VGI (WM276) molecular genotypes. Although R265 cna1Δ mutants were more tolerant to growth on solid medium at 37° compared with cna1Δ mutants generated from C. gattii R272, WM276, and C. neoformans H99 strains  compared with R265 wild-type in the presence of FK506  , suggesting divergent roles of calcineurin for controlling osmotic responses between Cryptococcus species and C. glabrata.To test whether loss of calcineurin function exerts a fungicidal effect at 37°, we shifted cells to 24° after exposure at 37°. We found that most calcineurin mutant cells from e viable . PreviouC. gattii outbreak isolate R265 compared with the C. gattii R272 and WM276 or C. neoformans H99 isolates  of wild-type and calcineurin mutants from three cna1 mutants exhibit severe growth defects at 37°, this phenotype led us to isolate (1) CNA1 complemented strains and (2) suppressed mutants via screening surviving colonies at a stringent cna1 non-permissive condition (38°) after biolistic transformation. We used the R272 CNA1 ORF flanked by ~1 kb of the 5′ NCR and 3′ NCR regions  to complement the cna1 mutation in strain YL165 and isolated three types of transformants able to grow at 38°, including: *1) CNA1 native-locus integration , (2) CNA1 ectopic integration , and (3) suppressor mutations  .Because R272 cna1 mutation were fully rescued on YPD medium containing SDS or fluconazole, while ectopic complementation (strain YC879) partially rescued the growth defects of the cna1 mutant on YPD medium containing SDS, but not fluconaole . On the other hand, suppressor mutants YC883 and YC886 were only rescued for the growth defects conferred by the cna1 mutation at 38° but not on YPD medium containing SDS or fluconazole. This suppressor analysis suggests a possible bifurcation of the calcineurin pathway, with one branch responding to temperature stress and another to cell wall/membrane stress. To decipher the nature of these suppressor mutations, we plan to conduct whole genome sequencing in the future.Although all three were restored to growth at 37°, the three types of transformants exhibited different responses to SDS and fluconazole. In the native-locus complemented strain YC875 the growth defects conferred by the C. gattii virulence in a murine inhalation model. Previous studies have shown that C. neoformans calcineurin is required for virulence in both rabbit and murine models  is more virulent than both WM276  and R272 , whereas WM276 is more virulent than the R272 strain . This finding suggests that a C. gattii environmental strain (WM276) can be as virulent as clinical isolates (R265 and R272). Because C. gattii calcineurin mutants from the three wild-type strains exhibit different responses to temperature, it was of interest to determine whether these mutants are still able to cause infection in a murine inhalation model. We found that C. gattii calcineurin mutants from all three molecular genotype isolates were avirulent or strongly attenuated for virulence .The strongly attenuated virulence of calcineurin mutants might be due to reduced or loss of C. gattii calcineurin is required for melanin and capsule production. We found that C. gattii calcineurin played no roles in melanin production , but minor roles in capsule production .Histopathological mucicarmine staining of tissues revealed that the wild-type R265, R272, and WM276 strains proliferated extensively in lung tissues, whereas calcineurin mutants from each wild type were not observed   , a reagewith SDS .et al. demonstrated that C. neoformans calcineurin (Cna1) interacts with the COPI component Sec28 and the COPII component Sec13 , suggesting that FK506 shows synergistic antifungal activity with fluconazole via a mechanism that is independent of the FK506 target proteins calcineurin and FKBP12  plus flucytosine  in four divided doses for at least 2 wk, followed by fluconazole  for a minimum of 8 wk  for thermotolerance and virulence in a murine inhalation model. Although calcineurin mutants from C. gattii R265 are more tolerant to growth at 37° , C. dubliniensis , C. lusitaniae (serum growth), A. fumigatus (filamentous growth), and M. oryzae  . Using a wax moth infection model incubated at 24°, we demonstrated that C. gattii calcineurin is required for virulence in the R265 and WM276 strain background , indicating that calcineurin also employs a mechanism other than thermotolerance for pathogenicity in the wax moth model. Interestingly, in the C. gattii R272 background calcineurin is only required for virulence in murine inhalation (37°), but not in a wax moth model (24°) , indicating that in C. gattii R272 the role of calcineurin in thermotolerance may be the major mechanism promoting infection in the murine inhalation model. However, the fact that the overall level of virulence of the R272 strain is reduced in the wax moth is a caveat to this conclusion.Because calcineurin mutants have pleotropic phenotypes linked to virulence, esponses . The funERG11, which is involved in ergosterol biosynthesis and cell membrane/wall integrity  exhibit different levels of azole tolerance. For example, R272 cna1Δ mutants are hypersensitive to fluconazole compared with R265 and WM272 cna1Δ mutants (cna1Δ mutants exhibit hypersensitivity to cell membrane/wall disturbing agents (cna1Δ mutation may result in azole hypersensitivity.Azoles target fungal 14α-demethylase encoded by  mutants . Becauseg agents  that is g agents , we suggC. gattii calcineurin promotes thermotolerance and other mechanisms during infection in both the murine inhalation and wax moth models, in addition to a requirement for calcineurin in azole tolerance of C. gattii. Therefore, our studies provide support for C. gattii calcineurin as an attractive target for developing calcineurin inhibitors that retain antifungal activity but are reduced for immunosuppressive activity.In summary, our data suggest that in