Search

Rift Valley fever (RVF) is a zoonotic disease of public health and economic importance. Uganda has reported sporadic outbreaks of RVF in both humans and animals across the country, especially in the southwestern part of the “cattle corridor” through an established viral hemorrhagic fever surveillance system. We report 52 human cases of laboratory-confirmed RVF from 2017 to 2020. The case fatality rate was 42%. Among those infected, 92% were males and 90% were adults (≥ 18 years). Clinical symptoms were characterized by fever (69%), unexplained bleeding (69%), headache (51%), abdominal pain (49%), and nausea and vomiting (46%). Most of the cases (95%) originated from central and western districts that are part of the cattle corridor of Uganda, where the main risk factor was direct contact with livestock (P = 0.009). Other predictors of RVF positivity were determined to be male gender (P = 0.001) and being a butcher (P = 0.04). Next-generation sequencing identified the predominant Ugandan clade as Kenya-2, observed previously across East Africa. There is need for further investigation and research into the effect and spread of this neglected tropical disease in Uganda and the rest of Africa. Control measures such as promoting vaccination and limiting animal–human transmission could be explored to reduce the impact of RVF in Uganda and globally.

Introduction Ebola virus (EBOV), species Zaire ebolavirus, may persist in the semen of male survivors of Ebola virus disease (EVD). We conducted a study of male survivors of the 2014–2016 EVD outbreak in Liberia and evaluated their immune responses to EBOV. We report here findings from the serologic testing of blood for EBOV-specific antibodies, molecular testing for EBOV in blood and semen, and serologic testing of peripheral blood mononuclear cells (PBMCs) in a subset of study participants. Methods We tested for EBOV RNA in blood by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and for anti-EBOV-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA) for 126 study participants. We performed PBMC analysis on a subgroup of 26 IgG-negative participants. Results All 126 participants tested negative for EBOV RNA in blood by qRT-PCR. The blood of 26 participants tested negative for EBOV-specific IgG antibodies by ELISA. PBMCs were collected from 23/26 EBOV IgG-negative participants. Of these, 1/23 participants had PBMCs that produced anti-EBOV-specific IgG antibodies upon stimulation with EBOV-specific glycoprotein (GP) and nucleoprotein (NP) antigens. Conclusions The blood of EVD survivors, collected when they did not have symptoms meeting the case definition for acute or relapsed EVD, is unlikely to pose a risk for EBOV transmission. We identified 1 IgM/IgG negative participant who had PBMCs that produced anti-EBOV-specific antibodies upon stimulation. Immunogenicity following acute EBOV infection may exist along a spectrum, and absence of antibody response should not be exclusionary in determining an individual’s status as a survivor of EVD.

Background Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola virus disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. Methods A cohort of 131 male EVD survivors in Liberia were enrolled in a case-case study. “Early clearers” were defined as those with 2 consecutive negative EBOV semen test results by real-time reverse-transcription polymerase chain reaction (rRT-PCR) ≥2 weeks apart within 1 year after discharge from the Ebola treatment unit or acute EVD. “Late clearers” had detectable EBOV RNA by rRT-PCR >1 year after discharge from the Ebola treatment unit or acute EVD. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical examinations and blood work. Results Compared with early clearers, late clearers were older (median, 42.5 years; P < .001) and experienced fewer severe clinical symptoms (median 2, P = .006). Late clearers had more lens opacifications (odds ratio, 3.9 [95% confidence interval, 1.1–13.3]; P = .03), after accounting for age, higher total serum immunoglobulin G3 (IgG3) titers (P = .005), and increased expression of the HLA-C*03:04 allele (0.14 [.02–.70]; P = .007). Conclusions Older age, decreased illness severity, elevated total serum IgG3 and HLA-C*03:04 allele expression may be risk factors for the persistence of EBOV in the semen of EVD survivors. EBOV persistence in semen may also be associated with its persistence in other immunologically protected sites, such as the eye.

The epidemiology of Rift Valley fever virus (RVFV) and Crimean-Congo hemorrhagic fever virus (CCHFV) in Jordan is unknown. Our investigation showed 3% of 989 tested dairy cattle, sheep, and goats were RVFV seropositive and 14% were CCHFV seropositive. Ongoing surveillance is needed to assess risk to humans and protect public health.

Background: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola Virus Disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to better understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. Methods: In 2017, a cohort of 131 male survivors of the 2014-2016 outbreak of EVD in Liberia were recruited from a national semen testing program into this case-case study. “Early clearers” were defined as those with two consecutive negative EBOV semen tests by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) at least two weeks apart within 1 year after discharge from the Ebola Treatment Unit (ETU). “Late clearers” had detectable EBOV RNA by rRT-PCR more than one year after ETU discharge. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical exams and blood work including chemistries, blood counts, hemoglobinopathies, human leukocyte antigen typing, and immunologic/inflammatory markers. Findings: Among 131 participants, 30 demonstrated long-term detection of EBOV RNA in semen by rRT-PCR, up to 852 days after ETU discharge. Compared to early clearers, late clearers were more likely to be older (median 42.5 years, p=0.0001) and reported having experienced fewer severe clinical symptoms (median 2, p=0.006). Late clearers were found to more frequently have lens opacifications (OR 3.9, 95%CI 1.1-13.3, p=0.03), after accounting for age. Finally, late clearers also exhibited elevated total serum IgG3 titers (p=0.007) and increased expression of the HLA-C*03:04 allele (OR 0.14, 95% CI 0.02-0.70, p=0.007). Interpretation: Older age and decreased illness severity may play a significant role in determining the clearance of EBOV from the semen of survivors. Immune and host genetic factors including total serum IgG3 and HLA-C*03:04 may mediate that interaction. In addition, our finding that late clearers were more likely to have lens opacifications after accounting for age suggests that individuals with EBOV persistence in semen may also have EBOV persistence in other immunologically protected sites, such as the eye. Identifying risk factors for viral persistence in semen among male survivors is critical to inform screening efforts and prevent recurrent outbreaks of EBOV. Funding Information: This was work was supported by funds from the CDC Foundation and the Centers for Disease Control and Prevention. This project has also been funded in whole or in part with federal funds from the Frederick National Laboratory for Cancer Research, under Contract No. HHSN261200800001E. Declaration of Interests: The authors report no competing interests. Ethics Approval Statement: The study protocol was approved by the CDC and Liberia MoH Institutional Review Boards and Meharry Medical College (for hemoglobinopathy testing).

To explore individual identity narratives of accommodation and resistance in relationship to dominant American social, political and cultural constructs, this paper uses the Listening Guide Method of Qualitative Inquiry (Gilligan et al., 2006) to investigate the intersectionality of race, ethnicity, gender and American identity during and post college among four second-generation, college educated, Korean American women. The analysis, drawing from the emergence of themes across interviews, found that participant women accommodated and/or resisted dominant American social, political, and cultural constructs in service of their individual Korean American identities narratives during and post college.

We report a case of infection with New York orthohantavirus in a woman who showed renal impairment and hemorrhage, complicated by hydrocephalus, in Long Island, New York, USA. Phylogenetic analysis showed that this virus was genetically similar to a New York orthohantavirus isolated in the same region during 1993.

The Democratic Republic of the Congo (DRC) declared an Ebola virus disease (EVD) outbreak in North Kivu in August 2018. By June 2019, the outbreak had spread to 26 health zones in northeastern DRC, causing >2,000 reported cases and >1,000 deaths. On June 10, 2019, three members of a Congolese family with EVD-like symptoms traveled to western Uganda’s Kasese District to seek medical care. Shortly thereafter, the Viral Hemorrhagic Fever Surveillance and Laboratory Program (VHF program) at the Uganda Virus Research Institute (UVRI) confirmed that all three patients had EVD. The Ugandan Ministry of Health declared an outbreak of EVD in Uganda’s Kasese District, notified the World Health Organization, and initiated a rapid response to contain the outbreak. As part of this response, UVRI and the United States Centers for Disease Control and Prevention, with the support of Uganda’s Public Health Emergency Operations Center, the Kasese District Health Team, the Superintendent of Bwera General Hospital, the United States Department of Defense’s Makerere University Walter Reed Project, and the United States Mission to Kampala’s Global Health Security Technical Working Group, jointly established an Ebola Field Laboratory in Kasese District at Bwera General Hospital, proximal to an Ebola Treatment Unit (ETU). The laboratory consisted of a rapid containment kit for viral inactivation of patient specimens and a GeneXpert Instrument for performing Xpert Ebola assays. Laboratory staff tested 76 specimens from alert and suspect cases of EVD; the majority were admitted to the ETU (89.3%) and reported recent travel to the DRC (58.9%). Although no EVD cases were detected by the field laboratory, it played an important role in patient management and epidemiological surveillance by providing diagnostic results in, Author summary Following an imported outbreak of Ebola virus disease in Uganda’s western Kasese District, the Uganda Virus Research Institute and the United States Centers for Disease Control and Prevention jointly established a frontline field laboratory to test specimens collected from alert and suspect cases for Ebola virus disease. Using a single room equipped with a rapid containment kit to safely inactivate patient specimens and a GeneXpert to perform the Xpert Ebola Assay, the field laboratory rapidly ruled-out Ebola virus disease as the cause of illness in 76 patients during its 46 operational days. All specimens were also referred to Uganda Virus Research Institute (Entebbe) for confirmatory Ebola virus testing and testing against a panel of viruses known to cause hemorrhagic fever in Uganda, in line with the National Viral Hemorrhagic Fever Program’s testing protocol and mandate. The Ebola field laboratory served as a valuable asset in the outbreak response by supporting patient management and epidemiological surveillance.

The genus Elizabethkingia is genetically heterogeneous, and the phenotypic similarities between recognized species pose challenges in correct identification of clinically derived isolates. In addition to the type species Elizabethkingia meningoseptica, and more recently proposed Elizabethkingia miricola, Elizabethkingia anophelis and Elizabethkingia endophytica, four genomospecies have long been recognized. By comparing historic DNA–DNA hybridization results with whole genome sequences, optical maps, and MALDI-TOF mass spectra on a large and diverse set of strains, we propose a comprehensive taxonomic revision of this genus. Genomospecies 1 and 2 contain the type strains E. anophelis and E. miricola, respectively. Genomospecies 3 and 4 are herein proposed as novel species named as Eliza-bethkingia bruuniana sp. nov. (type strain, G0146T = DSM 2975T = CCUG 69503T = CIP 111191T) and Elizabethkingia ursingii sp. nov. (type strain, G4122T = DSM 2974T = CCUG 69496T = CIP 111192T), respectively. Finally, the new species Elizabethkingia occulta sp. nov. (type strain G4070T = DSM 2976T = CCUG 69505T = CIP 111193T), is proposed.

BackgroundDuring 2017, a multistate outbreak investigation occurred after the confirmation of Seoul virus (SEOV) infections in people and pet rats. A total of 147 humans and 897 rats were tested.MethodsIn addition to immunoglobulin (Ig)G and IgM serology and traditional reverse-transcription polymerase chain reaction (RT-PCR), novel quantitative RT-PCR primers/probe were developed, and whole genome sequencing was performed.ResultsSeventeen people had SEOV IgM, indicating recent infection; 7 reported symptoms and 3 were hospitalized. All patients recovered. Thirty-one facilities in 11 US states had SEOV infection, and among those with ≥10 rats tested, rat IgG prevalence ranged 2%–70% and SEOV RT-PCR positivity ranged 0%–70%. Human laboratory-confirmed cases were significantly associated with rat IgG positivity and RT-PCR positivity (P = .03 and P = .006, respectively). Genomic sequencing identified >99.5% homology between SEOV sequences in this outbreak, and these were >99% identical to SEOV associated with previous pet rat infections in England, the Netherlands, and France. Frequent trade of rats between home-based ratteries contributed to transmission of SEOV between facilities.ConclusionsPet rat owners, breeders, and the healthcare and public health community should be aware and take steps to prevent SEOV transmission in pet rats and to humans. Biosecurity measures and diagnostic testing can prevent further infections.

Seoul orthohantavirus (SEOV) infections, uncommonly reported in the United States, often result in mild illness. We report a case of hemophagocytic lymphohistiocytosis secondary to SEOV infection that was domestically acquired in Washington, DC.

Marburg virus (MARV) causes sporadic outbreaks of severe Marburg virus disease (MVD). Most MVD outbreaks originated in East Africa and field studies in East Africa, South Africa, Zambia, and Gabon identified the Egyptian rousette bat (ERB; Rousettus aegyptiacus) as a natural reservoir. However, the largest recorded MVD outbreak with the highest case–fatality ratio happened in 2005 in Angola, where direct spillover from bats was not shown. Here, collaborative studies by the Centers for Disease Control and Prevention, Njala University, University of California, Davis USAID-PREDICT, and the University of Makeni identify MARV circulating in ERBs in Sierra Leone. PCR, antibody and virus isolation data from 1755 bats of 42 species shows active MARV infection in approximately 2.5% of ERBs. Phylogenetic analysis identifies MARVs that are similar to the Angola strain. These results provide evidence of MARV circulation in West Africa and demonstrate the value of pathogen surveillance to identify previously undetected threats., Egyptian rousette bats (ERBs) are natural reservoirs for Marburg virus (MARV), but these bats have not been linked to the MARV Angola strain that caused the largest and deadliest outbreak on record. Here, Amman et al., in a multi-institutional surveillance effort, identify and isolate Angola-like MARV in ERBs in West Africa.

Introduction In October 2017, a blood sample from a resident of Kween District, Eastern Uganda, tested positive for Marburg virus. Within 24 hour of confirmation, a rapid outbreak response was initiated. Here, we present results of epidemiological and laboratory investigations. Methods A district task force was activated consisting of specialised teams to conduct case finding, case management and isolation, contact listing and follow up, sample collection and testing, and community engagement. An ecological investigation was also carried out to identify the potential source of infection. Virus isolation and Next Generation sequencing were performed to identify the strain of Marburg virus. Results Seventy individuals (34 MVD suspected cases and 36 close contacts of confirmed cases) were epidemiologically investigated, with blood samples tested for MVD. Only four cases met the MVD case definition; one was categorized as a probable case while the other three were confirmed cases. A total of 299 contacts were identified; during follow- up, two were confirmed as MVD. Of the four confirmed and probable MVD cases, three died, yielding a case fatality rate of 75%. All four cases belonged to a single family and 50% (2/4) of the MVD cases were female. All confirmed cases had clinical symptoms of fever, vomiting, abdominal pain and bleeding from body orifices. Viral sequences indicated that the Marburg virus strain responsible for this outbreak was closely related to virus strains previously shown to be circulating in Uganda. Conclusion This outbreak of MVD occurred as a family cluster with no additional transmission outside of the four related cases. Rapid case detection, prompt laboratory testing at the Uganda National VHF Reference Laboratory and presence of pre-trained, well-prepared national and district rapid response teams facilitated the containment and control of this outbreak within one month, preventing nationwide and global transmission of the disease., Author summary Marburg virus disease (MVD) is caused by the virus that belongs to the same family as that of Ebola Virus disease. The disease is characterized by severe clinical symptoms such as high fever, diarrhoea and vomiting, and severe bleeding from most body openings. On average, 54% of the people who get infected with Marburg virus die from it [1]. In October 2017, Uganda reported an outbreak of MVD in the eastern district of Kween that borders Kenya in the Mount Elgon plains. This was the first time MVD was being detected in this part of the country since previous MVD outbreaks had been reported in the western part of Uganda. The Ministry of Health together with partners instituted rapid outbreak response for control. Investigations revealed a cluster of one family involving four cases whereby only one survived. The outbreak was traced back into bat caves in Kween district. Rock salt mining in the cave led to a spill-over of the virus into the human population with the subsequent person-to-person transmission. Through contact tracing and isolation of the infected people among other approaches, the outbreak was brought under control as explained in this article.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods.

Ninety-seven animal, human, and dairy Streptococcus porcinus or Streptococcus pseudoporcinus isolates in the CDC Streptococcus strain collection were evaluated on the basis of DNA-DNA reassociation, 16S rRNA and rpoB gene sequencing, conventional biochemical and Rapid ID 32 Strep identification methods, and antimicrobial susceptibility testing to determine their taxonomic status, characteristics for species differentiation, antimicrobial susceptibility, and relevance of clinical source. Nineteen of the 97 isolates (1 human, 18 swine) were identified as S. porcinus. The remaining 72 human isolates and 6 dairy isolates were identified as S. pseudoporcinus. The use of 16S rRNA or rpoB gene sequencing was required to differentiate S. porcinus from S. pseudoporcinus. The human and dairy S. pseudoporcinus isolates were biochemically distinct from each other as well as distinct by 16S rRNA and rpoB gene sequencing. Therefore, we propose the subspecies denominations S. pseudoporcinus subsp. hominis subsp. nov. for the human isolates and S. pseudoporcinus subsp. lactis subsp. nov. for the dairy isolates. Most strains were susceptible to the antimicrobials tested, with the exception of tetracycline. Two strains of each species were also resistant to clindamycin and erythromycin and carried the erm(A) (S. pseudoporcinus) or the erm(B) (S. porcinus) gene. S. porcinus was identified from a single human isolate recovered from a wound in an abattoir worker. S. pseudoporcinus was primarily isolated from the genitourinary tract of women but was also associated with blood, placental, and wound infections. Isolates reacting with group B antiserum and demonstrating wide beta-hemolysis should be suspected of being S. pseudoporcinus and not S. agalactiae.

Draft genome sequences of Elizabethkingia meningoseptica and representatives of each of its four historically described genomospecies were sequenced here. Preliminary analysis suggests that Elizabethkingia miricola belongs to genomospecies 2, and both Elizabethkingia anophelis and Elizabethkingia endophytica are most similar to genomospecies 1.

Ninety-seven animal, human, and dairy Streptococcus porcinus or Streptococcus pseudoporcinus isolates in the CDC Streptococcus strain collection were evaluated on the basis of DNA-DNA reassociation, 16S rRNA and rpoB gene sequencing, conventional biochemical and Rapid ID 32 Strep identification methods, and antimicrobial susceptibility testing to determine their taxonomic status, characteristics for species differentiation, antimicrobial susceptibility, and relevance of clinical source. Nineteen of the 97 isolates (1 human, 18 swine) were identified as S. porcinus . The remaining 72 human isolates and 6 dairy isolates were identified as S. pseudoporcinus. The use of 16S rRNA or rpoB gene sequencing was required to differentiate S. porcinus from S. pseudoporcinus . The human and dairy S. pseudoporcinus isolates were biochemically distinct from each other as well as distinct by 16S rRNA and rpoB gene sequencing. Therefore, we propose the subspecies denominations S. pseudoporcinus subsp . hominis subsp. nov. for the human isolates and S. pseudoporcinus subsp. lactis subsp. nov. for the dairy isolates. Most strains were susceptible to the antimicrobials tested, with the exception of tetracycline. Two strains of each species were also resistant to clindamycin and erythromycin and carried the erm (A) ( S. pseudoporcinus ) or the erm (B) ( S. porcinus ) gene. S. porcinus was identified from a single human isolate recovered from a wound in an abattoir worker. S. pseudoporcinus was primarily isolated from the genitourinary tract of women but was also associated with blood, placental, and wound infections. Isolates reacting with group B antiserum and demonstrating wide beta-hemolysis should be suspected of being S. pseudoporcinus and not S. agalactiae .

Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ∼10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.

Proper protein folding is key to producing recombinant proteins for structure determination. We have examined the effect of misfolded recombinant protein on gene expression in Escherichia coli. Comparison of expression patterns indicates a unique set of genes responding to translational misfolding. The response is in part analogous to heat shock and suggests a translational component to the regulation. We have further utilized the expression information to generate reporters responsive to protein misfolding. These reporters were used to identify properly folded recombinant proteins and to create soluble domains of insoluble proteins for structural studies.

The first monoclonal antibody was appproved for human clinical use in 1985. This antibody, OKT3 (Kung et al., 1979), was derived from a mouse hybridoma and produced anti-mouse reactions that limited its use, and hindered further development of monoclonal antibodies. Humanization technology eliminated these cross-species issues and resulted in the approval of the first recombinant “chimeric” antibody rituximab and “humanized” antibody trastuzumab in 1997 and 1998, respectively. The approval of these molecules opened the floodgates for antibody development, as over 20 antibodies are currently approved by the FDA for therapeutic use and hundreds are in clinical development (nelson et al., yEAR?). The need for humanized recombinant antibodies spurred the develoment of several technologies for their discovery and engineering.

Streptomyces cacaoi subsp. cacaoi , a Gram-positive, branching filamentous bacteria, was isolated from a scalp infection in a patient from Pondicherry, India. Phenotypic tests identified the isolate as a Streptomyces species, but 16S rRNA sequence analysis provided the species identification required for tracking of this emerging pathogen.

Despite the large sequence diversity present in the proteomes of known organisms, most functional proteins characterized to date assume one of relatively few distinct folds1-5, suggesting that nature uses a limited number of stable, soluble folds relative to what is theoretically possible in protein sequence space. These folds likely represent the divergent products of a limited set of ancient protein folds. However, protein sequences that fold into stable, unique topologies but are not encoded by any sequenced genome may also exist. It may be possible to identify other stable folds that simply have not yet been sampled in the course of evolution by means of an artificial selection and molecular evolution process. Methods such as DNA shuffling6 which mimic the combinatorial diversity mechanism of the immune system are among the most efficient methods to modify or enhance protein activity. Unfortunately, the structural diversity generated in a library of shuffled homologues is relatively small7-11, although newer methods for random fragment assembly that overcome sequence homology requirements may lead to libraries with increased structural diversity12,13. Alternatively, natural or in vitro combinatorial assembly of distinct protein subunits (e.g., subdomains, exons, etc.) can create significant structural and functional diversity14-19. For example, the mammalian blood clotting proteins plasminogen, protein C, urokinase and prothrombin are all derived from different combinations of 5 exons14. Sequence and structural studies also suggest that many existing TIM barrel proteins evolved from the combinatorial assembly of subunits from more primitive eightfold βα barrels15,16. Other approaches for creating libraries of novel sequences that fold into native-like protein structures include the use of binary patterned residues designed around an existing known fold and the random assembly of cassettes composed of as few as three amino acid residues17,18. A more recent report details the use of cassettes of binary patterned residues around known protein motifs that are ultimately randomly assembled without fitting any designed folding constraint19. However, these latter methods have not yet yielded stable, novel folds under physiological conditions20. Despite the considerable effort to identify or evolve new folds, only a limited number of distinct protein folds have been identified to date18. Here we describe a system for the synthesis of polypeptides with potentially novel folds in which existing sequence and structural data from bacterial proteins are used to assemble a pool of secondary structural elements (α-helices, β-strands and loops). These secondary structural elements are in turn recombined into a library of de novo sequences that may be screened or selected for folded proteins. Proteins that fold into a native state acquire measurable biophysical characteristics such as solubility, stable secondary structure, well-dispersed 1D NMR spectra indicative of packed side chains, and reversible two-state unfolding behavior. In this work, we chose to initially screen the library for polypeptides that are soluble in aqueous solution using a GFP fusion protein reporter that had been previously reported21. Indeed, several sequences with stable secondary structure are identified.

Recently, tRNA aminoacyl-tRNA synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl aminoacyl-tRNA synthetases that charge the unnatural amino acids p -bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substrate-bound WT synthetase, as well as a mutant synthetase that charges p -acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the α 8 -helix, which spans the WT active site. The high degree of structural plasticity that is observed in these aminoacyl-tRNA synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids.

It has been recently shown that orthogonal tRNA/aminoacyl-tRNA synthetase pairs can be evolved to allow genetic incorporation of unnatural amino acids into proteins in both prokaryotes and eukaryotes. Here we describe the crystal structure of an evolved aminoacyl-tRNA synthetase that charges the unnatural amino acid p-acetylphenylalanine. Molecular recognition is due to altered hydrogen bonding and packing interactions with bound substrate that result from changes in both side-chain and backbone conformation.