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During a disease outbreak, contact tracing and epidemiological analysis are of critical importance to analyze disease sources and transmission. To perform this epidemiological analysis, effective data visualization is necessary. In this study, the outbreak of COVID-19 was simulated within three metro Atlanta counties. Data was generated using contact tracing forms and used to create node and edge lists. Each node in the network contained a unique ID representing either a location or individual, as well as any contact tracing information related to each node. Data visualization was performed using MicrobeTrace, an online program developed by the CDC. Visualization of the contact tracing network enabled us to effectively analyze the transmission dynamics of SARS-CoV2. In the simulated network, a singular person node appeared to be linked to the most positive COVID-19 cases in the network. Similarly, a restaurant was identified as the place node with the greatest number of direct connections to positive persons, highlighting it as a potentially major source of exposure to SARS-CoV-2. This study illustrates the benefits of data visualization and demographic analysis using MicrobeTrace, which helps to target mitigation and prevention efforts, while also emphasizing the importance of contact tracing to reduce the transmission of disease.

Outbreak investigations use data from interviews, healthcare providers, laboratories and surveillance systems. However, integrated use of data from multiple sources requires a patchwork of software that present challenges in usability, interoperability, confidentiality, and cost. Rapid integration, visualization and analysis of data from multiple sources can guide effective public health interventions. We developed MicrobeTrace to facilitate rapid public health responses by overcoming barriers to data integration and exploration in molecular epidemiology. MicrobeTrace is a web-based, client-side, JavaScript application (https://microbetrace.cdc.gov) that runs in Chromium-based browsers and remains fully operational without an internet connection. Using publicly available data, we demonstrate the analysis of viral genetic distance networks and introduce a novel approach to minimum spanning trees that simplifies results. We also illustrate the potential utility of MicrobeTrace in support of contact tracing by analyzing and displaying data from an outbreak of SARS-CoV-2 in South Korea in early 2020. MicrobeTrace is developed and actively maintained by the Centers for Disease Control and Prevention. Users can email microbetrace@cdc.gov for support. The source code is available at https://github.com/cdcgov/microbetrace., Author summary Rapid advances in the fields of data science and bioinformatics have significantly improved molecular epidemiology tools used in public health and have led to major changes in the way outbreak investigation and pathogen transmission studies are conducted. However, the need for specialized computer skills often impedes the use of many of these tools in the public heath domain. We bridge this knowledge gap by development of an intuitive, standalone tool called MicrobeTrace to securely integrate, visualize and explore pathogen epidemiologic data. MicrobeTrace is an easy to use browser-based tool which can effectively merge contact tracing and/or microbial genomic data with demographic or behavioral information, resulting in elegant and informative networks as well as multiple customizable visualizations. MicrobeTrace can be used offline, with analyses being performed locally in the field, ensuring secure and confidential use of personally identifiable information (PII). We provide real world examples of how MicrobeTrace has been used in public health, including COVID outbreak investigations.

MotivationOutbreak investigations use data from interviews, healthcare providers, laboratories and surveillance systems. However, integrated use of data from multiple sources requires a patchwork of software that present challenges in usability, interoperability, confidentiality, and cost. Rapid integration, visualization and analysis of data from multiple sources can guide effective public health interventions.ResultsWe developed MicrobeTrace to facilitate rapid public health responses by overcoming barriers to data integration and exploration in molecular epidemiology. Using publicly available HIV sequences and other data, we demonstrate the analysis of viral genetic distance networks and introduce a novel approach to minimum spanning trees that simplifies results. We also illustrate the potential utility of MicrobeTrace in support of contact tracing by analyzing and displaying data from an outbreak of SARS-CoV-2 in South Korea in early 2020.Availability and ImplementationMicrobeTrace is a web-based, client-side, JavaScript application (https://microbetrace.cdc.gov) that runs in Chromium-based browsers and remains fully-operational without an internet connection. MicrobeTrace is developed and actively maintained by the Centers for Disease Control and Prevention. The source code is available athttps://github.com/cdcgov/microbetrace.Contactells@cdc.gov

The Democratic Republic of the Congo (DRC) has a history of nonhuman primate (NHP) consumption and exposure to simian retroviruses yet little is known about the extent of zoonotic simian retroviral infections in DRC. We examined the prevalence of human T-lymphotropic viruses (HTLV), a retrovirus group of simian origin, in a large population of persons with frequent NHP exposures and a history of simian foamy virus infection. We screened plasma from 3,051 persons living in rural villages in central DRC using HTLV EIA and western blot (WB). PCR amplification of HTLV tax and LTR sequences from buffy coat DNA was used to confirm infection and to measure proviral loads (pVLs). We used phylogenetic analyses of LTR sequences to infer evolutionary histories and potential transmission clusters. Questionnaire data was analyzed in conjunction with serological and molecular data. A relatively high proportion of the study population (5.4%, n = 165) were WB seropositive: 128 HTLV-1-like, 3 HTLV-2-like, and 34 HTLV-positive but untypeable profiles. 85 persons had HTLV indeterminate WB profiles. HTLV seroreactivity was higher in females, wives, heads of households, and increased with age. HTLV-1 LTR sequences from 109 persons clustered strongly with HTLV-1 and STLV-1 subtype B from humans and simians from DRC, with most sequences more closely related to STLV-1 from Allenopithecus nigroviridis (Allen’s swamp monkey). While 18 potential transmission clusters were identified, most were in different households, villages, and health zones. Three HTLV-1-infected persons were co-infected with simian foamy virus. The mean and median percentage of HTLV-1 pVLs were 5.72% and 1.53%, respectively, but were not associated with age, NHP exposure, village, or gender. We document high HTLV prevalence in DRC likely originating from STLV-1. We demonstrate regional spread of HTLV-1 in DRC with pVLs reported to be associated with HTLV disease, supporting local and national public health measures to prevent spread and morbidity., Author summary HTLV-1 is a human retrovirus of zoonotic simian origin that has spread globally causing inflammatory and carcinogenic disease. We previously showed that persons with high nonhuman primate (NHP) exposure in the Democratic Republic of the Congo (DRC) can be infected with another simian retrovirus, simian foamy virus (SFV), suggesting they are also at risk for infection with STLV-1. We conducted follow-up analysis of the same persons from rural villages of central DRC to determine exposure and transmission risks for STLV-1 and HTLV-1 infection. Most persons, especially women, reported high levels of NHP exposure. We identified possible introduction and spread of STLV-1 from a local monkey species across households, villages, and health zones in DRC. Most persons had HTLV-1 levels above those reported in previous studies for persons with disease. Our findings reveal that coordinated public health strategies are needed at both local and national levels to prevent further spread and morbidity of HTLV-1.

We obtained the full-length genome of a simian foamy virus (SFV) from an infected human. This virus originated from a baboon (Papio species, strain SFVpxx_hu9406). The genome is 13,113 nucleotides long with the canonical SFV genome structure. Phylogenetically, SFVpxx_hu9406 clustered closely with SFVpan_V909/03F from a captive baboon and other Cercopithecidae SFVs.

In 2014, the California Department of Public Health was notified by a local health department of a diagnosis of acute human immunodeficiency virus (HIV) infection* and rectal gonorrhea in a male adult film industry performer, aged 25 years (patient A). Patient A had a 6-day history of rash, fever, and sore throat suggestive of acute retroviral syndrome at the time of examination. He was informed of his positive HIV and gonorrhea test results 6 days after his examination. Patient A had a negative HIV-1 RNA qualitative nucleic acid amplification test (NAAT)(†) 10 days before symptom onset. This investigation found that during the 22 days between the negative NAAT and being informed of his positive HIV test results, two different production companies directed patient A to have condomless sex with a total of 12 male performers. Patient A also provided contact information for five male non-work-related sexual partners during the month before and after his symptom onset. Patient A had additional partners during this time period for which no locating information was provided. Neither patient A nor any of his interviewed sexual partners reported taking HIV preexposure prophylaxis (PrEP). Contact tracing and phylogenetic analysis of HIV sequences amplified from pretreatment plasma revealed that a non-work-related partner likely infected patient A, and that patient A likely subsequently infected both a coworker during the second film production and a non-work-related partner during the interval between his negative test and receipt of his positive HIV results. Adult film performers and production companies, medical providers, and all persons at risk for HIV should be aware that testing alone is not sufficient to prevent HIV transmission. Condom use provides additional protection from HIV and sexually transmitted infections (STIs). Performers and all persons at risk for HIV infection in their professional and personal lives should discuss the use of PrEP with their medical providers.

Over 40 species of nonhuman primates host simian immunodeficiency viruses (SIVs). In natural hosts, infection is generally assumed to be nonpathogenic due to a long coevolutionary history between host and virus, although pathogenicity is difficult to study in wild nonhuman primates. We used whole-blood RNA-seq and SIV prevalence from 29 wild Ugandan red colobus (Piliocolobus tephrosceles) to assess the effects of SIV infection on host gene expression in wild, naturally SIV-infected primates. We found no evidence for chronic immune activation in infected individuals, suggesting that SIV is not immunocompromising in this species, in contrast to human immunodeficiency virus in humans. Notably, an immunosuppressive gene, CD101, was upregulated in infected individuals. This gene has not been previously described in the context of nonpathogenic SIV infection. This expands the known variation associated with SIV infection in natural hosts and may suggest a novel mechanism for tolerance of SIV infection in the Ugandan red colobus.

Rapidly evolving RNA viruses continuously produce minority haplotypes that can become dominant if they are drug-resistant or can better evade the immune system. Therefore, early detection and identification of minority viral haplotypes may help to promptly adjust the patient's treatment plan preventing potential disease complications. Minority haplotypes can be identified using next-generation sequencing (NGS), but sequencing noise hinders accurate identification. The elimination of sequencing noise is a non-trivial task that still remains open. Here we propose CliqueSNV based on extracting pairs of statistically linked mutations from noisy reads. This effectively reduces sequencing noise and enables identifying minority haplotypes with the frequency below the sequencing error rate. We comparatively assess the performance of CliqueSNV using an in vitro mixture of nine haplotypes that were derived from the mutation profile of an existing HIV patient. We show that CliqueSNV can accurately assemble viral haplotypes with frequencies as low as 0.1% and maintains consistent performance across short and long bases sequencing platforms.

Isothermal nucleic acid amplification techniques, such as reverse-transcription loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are suitable for the development of a rapid, low-cost NAT that can be used at the POC. For demonstration of utility for global use, studies are needed to validate the performance of RT-LAMP for the detection of divergent subtypes. In this study, we designed and evaluated multiplexed HIV-1 integrase RT-LAMP primers to detect subtypes within group M, along with an RNase P positive internal processing and amplification control. Using a panel of 26 viral isolates representing the major circulating subtypes, we demonstrated detection of all isolates of subtypes A1, C, D, F1, F2, G, CRF01_AE, CRF02_AG, and two unique recombinant forms (URFs). A whole blood panel created with one representative isolate of each subtype was successfully amplified with the group M HIV-1 integrase and RNase P internal control primers. The group M HIV-1 RT-LAMP assay was further evaluated on 61 plasma specimens obtained from persons from Cameroon and Uganda. The sequence-conserved group M HIV-1 RT-LAMP primers, coupled to a low-cost amplification device, may improve diagnosis of acute infection at the POC and provide timely confirmation of HIV status.

Zoonotic pathogens cause an estimated 70% of emerging and re-emerging infectious diseases in humans. In sub-Saharan Africa, bushmeat hunting and butchering is considered the primary risk factor for human-wildlife contact and zoonotic disease transmission, particularly for the transmission of simian retroviruses. However, hunting is only one of many activities in sub-Saharan Africa that bring people and wildlife into contact. Here, we examine human-animal interaction in western Uganda, identifying patterns of injuries from animals and contact with nonhuman primates. Additionally, we identify individual-level risk factors associated with contact. Nearly 20% (246/1,240) of participants reported either being injured by an animal or having contact with a primate over their lifetimes. The majority (51.7%) of injuries were dog bites that healed with no long-term medical consequences. The majority (76.8%) of 125 total primate contacts involved touching a carcass; however, butchering (20%), hunting (10%), and touching a live primate (10%) were also reported. Red colobus (Piliocolobus rufomitratus tephrosceles) accounted for most primate contact events. Multivariate logistic regression indicated that men who live adjacent to forest fragments are at elevated risk of animal contact and specifically primate contact. Our results provide a useful comparison to West and Central Africa where "bushmeat hunting" is the predominant paradigm for human-wildlife contact and zoonotic disease transmission.

Of the seven known species of human retroviruses only one, human T-cell lymphotropic virus type 4 (HTLV-4), lacks a known animal reservoir. We report the largest screening for simian T-cell lymphotropic virus (STLV-4) to date in a wide range of captive and wild non-human primate (NHP) species from Cameroon. Among the 681 wild and 426 captive NHPs examined, we detected STLV-4 infection only among gorillas by using HTLV-4-specific quantitative polymerase chain reaction. The large number of samples analyzed, the diversity of NHP species examined, the geographic distribution of infected animals relative to the known HTLV-4 case, as well as detailed phylogenetic analyses on partial and full genomes, indicate that STLV-4 is endemic to gorillas, and that rather than being an ancient virus among humans, HTLV-4 emerged from a gorilla reservoir, likely through the hunting and butchering of wild gorillas. Our findings shed further light on the importance of gorillas as keystone reservoirs for the evolution and emergence of human infectious diseases and provide a clear course for preventing HTLV-4 emergence through management of human contact with wild gorillas, the development of improved assays for HTLV-4/STLV-4 detection and the ongoing monitoring of STLV-4 among gorillas and for HTLV-4 zoonosis among individuals exposed to gorilla populations.

In January 2015, an outbreak of undiagnosed human immunodeficiency virus (HIV) infections among persons who inject drugs (PWID) was recognized in rural Indiana. By September 2016, 205 persons in this community of approximately 4400 had received a diagnosis of HIV infection. We report results of new approaches to analyzing epidemiologic and laboratory data to understand transmission during this outbreak. HIV genetic distances were calculated using the polymerase region. Networks were generated using data about reported high-risk contacts, viral genetic similarity, and their most parsimonious combinations. Sample collection dates and recency assay results were used to infer dates of infection. Epidemiologic and laboratory data each generated large and dense networks. Integration of these data revealed subgroups with epidemiologic and genetic commonalities, one of which appeared to contain the earliest infections. Predicted infection dates suggest that transmission began in 2011, underwent explosive growth in mid-2014, and slowed after the declaration of a public health emergency. Results from this phylodynamic analysis suggest that the majority of infections had likely already occurred when the investigation began and that early transmission may have been associated with sexual activity and injection drug use. Early and sustained efforts are needed to detect infections and prevent or interrupt rapid transmission within networks of uninfected PWID.

Voltage-gated sodium channels (VGSCs) are essential for the generation and propagation of action potentials in electrically excitable cells. Dominant mutations in SCN1A, which encodes the Nav1.1 VGSC α-subunit, underlie several forms of epilepsy, including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Electrophysiological analyses of DS and GEFS+ mouse models have led to the hypothesis that SCN1A mutations reduce the excitability of inhibitory cortical and hippocampal interneurons. To more directly examine the relative contribution of inhibitory interneurons and excitatory pyramidal cells to SCN1A-derived epilepsy, we first compared the expression of Nav1.1 in inhibitory parvalbumin (PV) interneurons and excitatory neurons from P22 mice using fluorescent immunohistochemistry. In the hippocampus and neocortex, 69% of Nav1.1 immunoreactive neurons were also positive for PV. In contrast, 13% and 5% of Nav1.1 positive cells in the hippocampus and neocortex, respectively, were found to co-localize with excitatory cells identified by CaMK2α immunoreactivity. Next, we reduced the expression of Scn1a in either a subset of interneurons (mainly PV interneurons) or excitatory cells by crossing mice heterozygous for a floxed Scn1a allele to either the Ppp1r2-Cre or EMX1-Cre transgenic lines, respectively. The inactivation of one Scn1a allele in interneurons of the neocortex and hippocampus was sufficient to reduce thresholds to flurothyl- and hyperthermia-induced seizures, whereas thresholds were unaltered following inactivation in excitatory cells. Reduced interneuron Scn1a expression also resulted in the generation of spontaneous seizures. These findings provide direct evidence for an important role of PV interneurons in the pathogenesis of Scn1a-derived epilepsies.

Increased HIV transmission in persons who inject drugs (PWIDs) has led to subepidemics and outbreaks in several countries in Europe, including Bulgaria. In this study in Bulgaria, we investigate the origin and spatiotemporal evolutionary history of HIV-1 infections in PWIDs and the distribution of antiretroviral resistance mutations and hepatitis co-infections in these populations. We analyzed HIV-1 polymerase sequences available from 117 of 359 PWIDs diagnosed with HIV/AIDS from 1999 to 2011. Of these, 50 (42.7%) were classified as CRF02_AG, 41 (35.0%) CRF01_AE, 12 (10.3%) URFs, ten (8.5%) subtype B, two (1.7%) subtype F1 and two (1.7%) CRF14_BG. Most recent common ancestor dating suggests that CRF01_AE was likely first introduced from Southeast Asia into persons reporting heterosexual infection in Bulgaria in 1992 and spread subsequently to PWIDs in the capital city of Sofia around 2003. Conversely, CRF02_AG in Bulgaria was likely first introduced into PWID from Germany in 2000 and later entered heterosexual populations around 2009. The overall prevalence of resistance mutations was 6.8% (8/117), of which 5.1% (5/117) was observed in patients on antiretroviral therapy and 1.7% (2/117) was from transmitted drug resistance mutations in drug-naive individuals. 189/204 (92.6%) PWIDs were also co-infected with hepatitis C (HCV) and 31/183 (16.9%) were co-infected with hepatitis B (HBV). Our study provides valuable molecular epidemiological information on the introduction and distribution of the main HIV-1 subtypes, resistance mutations and hepatitis co-infections among PWIDs with HIV-1 in Bulgaria which can be used to target prevention efforts.

Zoonotic transmission of simian retroviruses in West-Central Africa occurring in primate hunters has resulted in pandemic spread of human immunodeficiency viruses (HIVs) and human T-lymphotropic viruses (HTLVs). While simian foamy virus (SFV) and simian T- lymphotropic virus (STLV)-like infection were reported in healthy persons exposed to nonhuman primates (NHPs) in West-Central Africa, less is known about the distribution of these viruses in Western Africa and in hospitalized populations. We serologically screened for SFV and STLV infection using 1,529 specimens collected between 1985 and 1997 from Côte d'Ivoire patients with high HIV prevalence. PCR amplification and analysis of SFV, STLV, and HIV/SIV sequences from PBMCs was used to investigate possible simian origin of infection. We confirmed SFV antibodies in three persons (0.2%), two of whom were HIV-1-infected. SFV polymerase (pol) and LTR sequences were detected in PBMC DNA available for one HIV-infected person. Phylogenetic comparisons with new SFV sequences from African guenons showed infection likely originated from a Chlorocebus sabaeus monkey endemic to Côte d'Ivoire. 4.6% of persons were HTLV seropositive and PCR testing of PBMCs from 15 HTLV seroreactive persons identified nine with HTLV-1 and one with HTLV-2 LTR sequences. Phylogenetic analysis showed that two persons had STLV-1-like infections, seven were HTLV-1, and one was an HTLV-2 infection. 310/858 (53%), 8/858 (0.93%), and 18/858 (2.1%) were HIV-1, HIV-2, and HIV-positive but undifferentiated by serology, respectively. No SIV sequences were found in persons with HIV-2 antibodies (n = 1) or with undifferentiated HIV results (n = 7). We document SFV, STLV-1-like, and dual SFV/HIV infection in Côte d'Ivoire expanding the geographic range for zoonotic simian retrovirus transmission to West Africa. These findings highlight the need to define the public health consequences of these infections. Studying dual HIV-1/SFV infections in immunocompromised populations may provide a new opportunity to better understand SFV pathogenicity and transmissibility in humans.

© 2015 Elsevier Inc. Understanding the role of SCN8A in epilepsy and behavior is critical in light of recently identified human SCN8A epilepsy mutations. We have previously demonstrated that Scn8amedand Scn8amed-jomice carrying mutations in the Scn8a gene display increased resistance to flurothyl and kainic acid-induced seizures; however, they also exhibit spontaneous absence seizures. To further investigate the relationship between altered SCN8A function and epilepsy, we introduced the SCN1A-R1648H mutation, identified in a family with generalized epilepsy with febrile seizures plus (GEFS+), into the corresponding position (R1627H) of the mouse Scn8a gene. Heterozygous R1627H mice exhibited increased resistance to some forms of pharmacologically and electrically induced seizures and the mutant Scn8a allele ameliorated the phenotype of Scn1a-R1648H mutants. Hippocampal slices from heterozygous R1627H mice displayed decreased bursting behavior compared to wild-type littermates. Paradoxically, at the homozygous level, R1627H mice did not display increased seizure resistance and were susceptible to audiogenic seizures. We furthermore observed increased hippocampal pyramidal cell excitability in heterozygous and homozygous Scn8a-R1627H mutants, and decreased interneuron excitability in heterozygous Scn8a-R1627H mutants. These results expand the phenotypes associated with disruption of the Scn8a gene and demonstrate that an Scn8a mutation can both confer seizure protection and increase seizure susceptibility.

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability.

Background While simian foamy viruses have co-evolved with their primate hosts for millennia, most scientific studies have focused on understanding infection in Old World primates with little knowledge available on the epidemiology and natural history of SFV infection in New World primates (NWPs). To better understand the geographic and species distribution and evolutionary history of SFV in NWPs we extend our previous studies in Brazil by screening 15 genera consisting of 29 NWP species (140 monkeys total), including five genera (Brachyteles, Cacajao, Callimico, Mico, and Pithecia) not previously analyzed. Monkey blood specimens were tested using a combination of both serology and PCR to more accurately estimate prevalence and investigate transmission patterns. Sequences were phylogenetically analyzed to infer SFV and host evolutionary histories. Results The overall serologic and molecular prevalences were 42.8 and 33.6 %, respectively, with a combined assay prevalence of 55.8 %. Discordant serology and PCR results were observed for 28.5 % of the samples, indicating that both methods are currently necessary for estimating NWP SFV prevalence. SFV prevalence in sexually mature NWPs with a positive result in any of the WB or PCR assays was 51/107 (47.7 %) compared to 20/33 (61 %) for immature animals. Epidemiological analyses revealed an increase in SFV prevalence with age in captive Cebus monkeys. Phylogenetic analysis identified novel SFVs in Cacajao,Leontopithecus, and Chiropotes species that had 6–37 % nucleotide divergence to other NWP SFV. Comparison of host and SFV phylogenies showed an overall cospeciation evolutionary history with rare ancient and contemporaneous host-switching for Saimiri and Leontopithecus and Cebus xanthosternos, respectively. Conclusions We identified novel SFV in four neotropical monkey genera in Brazil and demonstrate that SFV prevalence increases with age in Cebus monkeys. Importantly, our test results suggest that both molecular and serological screening are currently required to accurately determine infection with NWP SFV. Our study significantly expands knowledge of the epidemiology and natural history of NWP SFVs. The tools and information provided in our study will facilitate further investigation of SFV in NWPs and the potential for zoonotic infection with these viruses.

BACKGROUND: Migrainous vertigo (MV) is increasingly recognized as a common cause of episodic vertigo. MV displays several clinical similarities with familial hemiplegic migraine (FHM) and episodic ataxia type 2 (EA-2), which have been linked to mutations in 3 genes, CACNA1A, encoding a neuronal calcium channel alpha subunit, ATP1A2, encoding a catalytic subunit of a Na(+)/K(+)-ATPase, and most recently the voltage-gated sodium channel SCN1A. The present study explored the hypothesis that mutations in CACNA1A, ATP1A2, SCN1A, and the calcium channel beta(4) subunit CACNB4 confer susceptibility to MV. METHODS: Mutation analysis of the coding exons and exon/intron junctions of CACNA1A, ATP1A2, SCN1A, and CACNB4 was performed in 14 unrelated MV patients by conformation sensitive gel electrophoresis and automated sequence analysis. RESULTS: Analysis of the 4 candidate genes in the 14 MV patients resulted in the identification of a total of 26 sequence variants. The silent substitution D29D in CACNB4 was observed in 2 MV patients and was not present in 46 ethnically matched control DNA samples. The remaining variants were also observed in control DNA samples and the allele frequencies of variants that resulted in amino acid substitutions were not significantly different between patients and controls. CONCLUSIONS: Based on this group of patients there is no evidence that the genes causing FHM and EA-2 represent major susceptibility loci for MV.

One of the most important processes to the survival of a species is its ability to reproduce. In plants, SEPALLATA-class MADS-box genes have been found to control the development of the inner whorls of flowers. However, while much is known about floral development in herbaceous plants, similar systems in woody trees remain poorly understood. Populus tremuloides (trembling aspen) is a widespread North American tree having important economic value, and its floral development differs from that of well-studied species in that the flowers have only two whorls and are truly unisexual. Sequence based analyses indicate that PTM3 (Populus tremuloides MADS-box 3), and a duplicate gene PTM4, are related to the SEPALLATA1-and 2-class of MADS-box genes. Another gene, PTM6, is related to SEP3, and each of these genes has a counterpart in the poplar genomic database along with additional members of the A, B, C, D, and E-classes of MADS-box genes. PTM3/4 and 6 are expressed in all stages of male and female aspen floral development. However, PTM3/4 is also expressed in the terminal buds, young leaves, and young stems. In situ RNA localization identified PTM3/4 and 6 transcripts predominantly in the inner, sexual whorl, within developing ovules of female flowers and anther primordia of male flowers. Tree researchers often use heterologous systems to help study tree floral development due to the long juvenile periods found in most trees. We found that the participation of PTM3/4 in floral development is supported by transgenic experiments in both P. tremuloides and heterologous systems such as tobacco and Arabidopsis. However, phenotypic artifacts were observed in the heterologous systems. Together the results suggest a role for poplar SEP-class genes in reproductive viability.

Background: Human immunodeficiency virus (HIV) type 1 and 2, the causative agents of acquired immunodeficiency syndrome (AIDS), emerged from African non-human primates (NHPs) through zoonotic transmission of simian immunodeficiency viruses (SIV). Among African NHPs, the Cercopithecus genus contains the largest number of species known to harbor SIV. However, our understanding of the diversity and evolution of SIVs infecting this genus is limited by incomplete taxonomic and geographic sampling, particularly in East Africa. In this study, we screened blood specimens from red-tailed guenons (Cercopithecus ascanius schmidti) from Kibale National Park, Uganda, for the presence of novel SIVs using unbiased deep-sequencing. Findings: We describe and characterize the first full-length SIV genomes from wild red-tailed guenons in Kibale National Park, Uganda. This new virus, tentatively named SIVrtg_Kib, was detected in five out of twelve animals and is highly divergent from other Cercopithecus SIVs as well as from previously identified SIVs infecting red-tailed guenons, thus forming a new SIV lineage. Conclusions: Our results show that the genetic diversity of SIVs infecting red-tailed guenons is greater than previously appreciated. This diversity could be the result of cross-species transmission between different guenon species or limited gene flow due to geographic separation among guenon populations.

The incidence of human immunodeficiency virus (HIV) infection has significantly increased among black men who have sex with men (MSM) in the United States, and young black MSM have been disproportionately affected. HIVinfected black MSM are also less likely to engage in HIV care and achieve viral suppression than MSM of other races/ethnicities. Engaging in care and achieving viral suppression is a multistep process that starts with diagnosis. Diagnosing persons unaware of their HIV status traditionally has been a critical component of HIV partner services, but partner services also provide an important opportunity to reengage HIVinfected partners in medical care. One approach for partner services involves contacting partners of persons with newly diagnosed HIV infection and using sexual and social network and molecular phylogenetic data to improve the continuum of HIV care among black MSM. To evaluate the effectiveness of that approach, results from a prospective partner services study conducted in North Carolina were examined, and one of the partner networks identified through this study was evaluated in depth. Overall, partner services were provided to 30 black, HIV-infected MSM who named 95 sex partners and social contacts, of whom 39 (41%) previously had been diagnosed with HIV infection. The partner network evaluation demonstrated that HIV-infected and HIV-negative partners were frequently in the same network, and that the majority of HIV-infected partners were already aware of their diagnosis but had not achieved viral suppression. Using partner services to ensure that HIV-infected partners are linked to care and treatment might reduce HIV transmission and might improve outcomes along the continuum of care.

Foamy viruses naturally infect a wide range of mammals, including Old World (OWP) and New World primates (NWP), which are collectively called simian foamy viruses (SFV). While NWP species in Central and South America are highly diverse, only SFV from captive marmoset, spider monkey, and squirrel monkey have been genetically characterized and the molecular epidemiology of SFV infection in NWPs remains unknown. We tested a large collection of genomic DNA (n = 332) comprising 14 genera of NWP species for the presence of SFV polymerase (pol) sequences using generic PCR primers. Further molecular characterization of positive samples was carried out by LTR-gag and larger pol sequence analysis. We identified novel SFVs infecting nine NWP genera. Prevalence rates varied between 14–30% in different species for which at least 10 specimens were tested. High SFV genetic diversity among NWP up to 50% in LTR-gag and 40% in pol was revealed by intragenus and intrafamilial comparisons. Two different SFV strains infecting two captive yellow-breasted capuchins did not group in species-specific lineages but rather clustered with SFVs from marmoset and spider monkeys, indicating independent cross-species transmission events. We describe the first SFV epidemiology study of NWP, and the first evidence of SFV infection in wild NWPs. We also document a wide distribution of distinct SFVs in 14 NWP genera, including two novel co-speciating SFVs in capuchins and howler monkeys, suggestive of an ancient evolutionary history in NWPs for at least 28 million years. A high SFV genetic diversity was seen among NWP, yet these viruses seem able to jump between NWP species and even genera. Our results raise concerns for the risk of zoonotic transmission of NWP SFV to humans as these primates are regularly hunted for food or kept as pets in forest regions of South America.

Limited information is available to describe the molecular epidemiology of HIV-1 in Bulgaria. To better understand the genetic diversity and the epidemiologic dynamics of HIV-1 we analyzed 125 new polymerase (pol) sequences from Bulgarians diagnosed through 2009 and 77 pol sequences available from our previous study from persons infected prior to 2007. Epidemiologic and demographic information was obtained from each participant and phylogenetic analysis was used to infer HIV-1 evolutionary histories. 120 (59.5%) persons were infected with one of five different HIV-1 subtypes (A1, B, C, F1 and H) and 63 (31.2%) persons were infected with one of six different circulating recombinant forms (CRFs; 01_AE, 02_AG, 04_cpx, 05_DF, 14_BG, and 36_cpx). We also for the first time identified infection with two different clusters of unique A-like and F-like sub-subtype variants in 12 persons (5.9%) and seven unique recombinant forms (3.5%), including a novel J/C recombinant. While subtype B was the major genotype identified and was more prevalent in MSM and increased between 2000-2005, most non-B subtypes were present in persons ≥45 years old. CRF01_AE was the most common non-B subtype and was higher in women and IDUs relative to other risk groups combined. Our results show that HIV-1 infection in Bulgaria reflects the shifting distribution of genotypes coincident with the changing epidemiology of the HIV-1 epidemic among different risk groups. Our data support increased public health interventions targeting IDUs and MSM. Furthermore, the substantial and increasing HIV-1 genetic heterogeneity, combined with fluctuating infection dynamics, highlights the importance of sustained and expanded surveillance to prevent and control HIV-1 infection in Bulgaria.

Background: The association of xenotropic murine leukemia virus (MLV)-related virus (XMRV) in prostate cancer and chronic fatigue syndrome reported in previous studies remains controversial as these results have been questioned by recent data. Nonetheless, concerns have been raised regarding contamination of human vaccines as a possible source of introduction of XMRV and MLV into human populations. To address this possibility, we tested eight live attenuated human vaccines using generic PCR for XMRV and MLV sequences. Viral metagenomics using deep sequencing was also done to identify the possibility of other adventitious agents. Results: All eight live attenuated vaccines, including Japanese encephalitis virus (JEV) (SA-14-14-2), varicella (Varivax), measles, mumps, and rubella (MMR-II), measles (Attenuvax), rubella (Meruvax-II), rotavirus (Rotateq and Rotarix), and yellow fever virus were negative for XMRV and highly related MLV sequences. However, residual hamster DNA, but not RNA, containing novel endogenous gammaretrovirus sequences was detected in the JEV vaccine using PCR. Metagenomics analysis did not detect any adventitious viral sequences of public health concern. Intracisternal A particle sequences closest to those present in Syrian hamsters and not mice were also detected in the JEV SA-14-14-2 vaccine. Combined, these results are consistent with the production of the JEV vaccine in Syrian hamster cells. Conclusions: We found no evidence of XMRV and MLV in eight live attenuated human vaccines further supporting the safety of these vaccines. Our findings suggest that vaccines are an unlikely source of XMRV and MLV exposure in humans and are consistent with the mounting evidence on the absence of these viruses in humans.

Background XMRV, a xenotropic murine leukemia virus (MuLV)-related virus, was recently identified by PCR testing in 67% of persons with chronic fatigue syndrome (CFS) and in 3.7% of healthy persons from the United States. To investigate the association of XMRV with CFS we tested blood specimens from 51 persons with CFS and 56 healthy persons from the US for evidence of XMRV infection by using serologic and molecular assays. Blinded PCR and serologic testing were performed at the US Centers for Disease Control and Prevention (CDC) and at two additional laboratories. Results Archived blood specimens were tested from persons with CFS defined by the 1994 international research case definition and matched healthy controls from Wichita, Kansas and metropolitan, urban, and rural Georgia populations. Serologic testing at CDC utilized a Western blot (WB) assay that showed excellent sensitivity to MuLV and XMRV polyclonal or monoclonal antibodies, and no reactivity on sera from 121 US blood donors or 26 HTLV-and HIV-infected sera. Plasma from 51 CFS cases and plasma from 53 controls were all WB negative. Additional blinded screening of the 51 cases and 53 controls at the Robert Koch Institute using an ELISA employing recombinant Gag and Env XMRV proteins identified weak seroreactivity in one CFS case and a healthy control, which was not confirmed by immunofluorescence. PCR testing at CDC employed a gag and a pol nested PCR assay with a detection threshold of 10 copies in 1 ug of human DNA. DNA specimens from 50 CFS patients and 56 controls and 41 US blood donors were all PCR-negative. Blinded testing by a second nested gag PCR assay at the Blood Systems Research Institute was also negative for DNA specimens from the 50 CFS cases and 56 controls. Conclusions We did not find any evidence of infection with XMRV in our U.S. study population of CFS patients or healthy controls by using multiple molecular and serologic assays. These data do not support an association of XMRV with CFS.

Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),(7) severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1a(RH/RH) mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1a(RH/+) heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1a(RH/+) and Scn1a(RH/RH) mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.

This thesis explores the high-performance implementation of a phase recovery algorithm for microstructure reconstruction of materials. Implementations on a variety of high-performance computing platforms, including multi-core and Graphics Processing Unit (GPU), were investigated and compared. The phase recovery algorithm is an iterative process requiring multiple Discrete Fourier Transform (DFT) computations each iteration. In order to achieve high-performance, it is necessary to use highly optimized fast Fourier transform (FFT) code to compute the DFTs. In our investigation, several FFT libraries, including FFTW, the Intel R Math Kernel Library (MKL), the CUFFT library for the NVIDIAR GPU, and the SPIRAL generated code, were used and compared. The SPIRAL system provides an extensible framework for generating and automatically optimizing implementations of DSP (digital signal processing) algorithms described using mathematical formulas, and is the most extensible of the platforms investigated here. The phas recovery algorithm intersperses FFT computations with point-wise computations, and while the FFTs are the dominant computation, the point-wise operations can have a signi cant impact on the overall performance. Therefore, simply relying on the performance of an optimized FFT library is insu cient to obtain optimal performance. Unlike the FFTW, MKL, and CUFFT libraries, the SPIRAL system allows the FFTs to be combined with the point-wise operations and the entire algorithm to be optimized. In this thesis, we obtained a mathematical formula representing the phase recovery algorithm that can be incorporated into the SPIRAL framework and utilize SPIRAL's parallel and vector code generation and optimization facilities. The SPIRAL code generated in this thesis is sequential. We estimate that with a vectorized and parallelized SPIRAL implementation, it is possible to obtain a 1.5-fold speedup for two-dimensional (2D) phase recovery and 1.88-fold speed up for 3D phase recovery over the MKL implementation.

Benign familial neonatal convulsions (BFNC) is an epileptic disorder caused by dominant mutations in the genes KCNQ2 and KCNQ3 encoding the K+ channels K(V)7.2 and K(V)7.3. We identified two novel KCNQ2 mutations in two BFNC families. One mutation predicted a truncated protein (S247X) that lacks the channel's pore region, the other resulted in the amino acid substitution S122L in the S2 segment of K(V)7.2. In comparison to wild-type (WT) K(V)7.2, functional analysis of S122L mutant channels in Xenopus oocytes revealed a significant positive shift and increased slope of the activation curve leading to significant current reduction in the subthreshold range of an action potential (75% reduction at -50 mV). Our results establish an important role of the K(V)7.2 S2 segment in voltage-dependent channel gating and demonstrate in a human disease that subthreshold voltages are likely to represent the physiologically relevant range for this K+ channel to regulate neuronal firing.

The xenotropic murine leukemia virus (MLV)-related viruses (XMRV) have been reported in persons with prostate cancer, chronic fatigue syndrome, and less frequently in blood donors. Polytropic MLVs have also been described in persons with CFS and blood donors. However, many studies have failed to confirm these findings, raising the possibility of contamination as a source of the positive results. One PCR reagent, Platinum Taq polymerase (pol) has been reported to contain mouse DNA that produces false-positive MLV PCR results. We report here the finding of a large number of PCR reagents that have low levels of MLV sequences. We found that recombinant reverse-transcriptase (RT) enzymes from six companies derived from either MLV or avian myeloblastosis virus contained MLV pol DNA sequences but not gag or mouse DNA sequences. Sequence and phylogenetic analysis showed high relatedness to Moloney MLV, suggesting residual contamination with an RT-containing plasmid. In addition, we identified contamination with mouse DNA and a variety of MLV sequences in commercially available human DNAs from leukocytes, brain tissues, and cell lines. These results identify new sources of MLV contamination and highlight the importance of careful pre-screening of commercial specimens and diagnostic reagents to avoid false-positive MLV PCR results.

Background Zoonotic transmission of simian retroviruses in Central Africa is ongoing and can result in pandemic human infection. While simian foamy virus (SFV) infection was reported in primate hunters in Cameroon and Gabon, little is known about the distribution of SFV in Africa and whether human-to-human transmission and disease occur. We screened 3,334 plasmas from persons living in rural villages in central Democratic Republic of Congo (DRC) using SFV-specific EIA and Western blot (WB) tests. PCR amplification of SFV polymerase sequences from DNA extracted from buffy coats was used to measure proviral loads. Phylogenetic analysis was used to define the NHP species origin of SFV. Participants completed questionnaires to capture NHP exposure information. Results Sixteen (0.5%) samples were WB-positive; 12 of 16 were from women (75%, 95% confidence limits 47.6%, 92.7%). Sequence analysis detected SFV in three women originating from Angolan colobus or red-tailed monkeys; both monkeys are hunted frequently in DRC. NHP exposure varied and infected women lived in distant villages suggesting a wide and potentially diverse distribution of SFV infections across DRC. Plasmas from 22 contacts of 8 WB-positive participants were all WB negative suggesting no secondary viral transmission. Proviral loads in the three women ranged from 14 – 1,755 copies/105 cells. Conclusions Our study documents SFV infection in rural DRC for the first time and identifies infections with novel SFV variants from Colobus and red-tailed monkeys. Unlike previous studies, women were not at lower risk for SFV infection in our population, providing opportunities for spread of SFV both horizontally and vertically. However, limited testing of close contacts of WB-positive persons did not identify human-to-human transmission. Combined with the broad behavioral risk and distribution of NHPs across DRC, our results suggest that SFV infection may have a wider geographic distribution within DRC. These results also reinforce the potential for an increased SFV prevalence throughout the forested regions of Africa where humans and simians co-exist. Our finding of endemic foci of SFV infection in DRC will facilitate longitudinal studies to determine the potential for person-to-person transmissibility and pathogenicity of these zoonotic retroviral infections.