Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

Ecogeographic Drivers of the Spatial Spread of Highly Pathogenic Avian Influenza Outbreaks in Europe and the United States, 2016-Early 2022.

H5Nx highly pathogenic avian influenza (HPAI) viruses of clade 2.3.4.4 have caused outbreaks in Europe among wild and domestic birds since 2016 and were introduced to North America via wild migratory birds in December 2021. We examined the spatiotemporal extent of HPAI viruses across continents and characterized ecological and environmental predictors of virus spread between geographic regions by constructing a Bayesian phylodynamic generalized linear model (phylodynamic-GLM). The findings demonstrate localized epidemics of H5Nx throughout Europe in the first several years of the epizootic, followed by a singular branching point where H5N1 viruses were introduced to North America, likely via stopover locations throughout the North Atlantic. Once in the United States (US), H5Nx viruses spread at a greater rate between US-based regions as compared to prior spread in Europe. We established that geographic proximity is a predictor of virus spread between regions, implying that intercontinental transport across the Atlantic Ocean is relatively rare. An increase in mean ambient temperature over time was predictive of reduced H5Nx virus spread, which may reflect the effect of climate change on declines in host species abundance, decreased persistence of the virus in the environment, or changes in migratory patterns due to ecological alterations. Our data provide new knowledge about the spread and directionality of H5Nx virus dispersal in Europe and the US during an actively evolving intercontinental outbreak, including predictors of virus movement between regions, which will contribute to surveillance and mitigation strategies as the outbreak unfolds, and in future instances of uncontained avian spread of HPAI viruses.

Comparing the transmission potential from sequence and surveillance data of 2009 North American influenza pandemic waves.

Technological advancements in phylodynamic modeling coupled with the accessibility of real-time pathogen genetic data are increasingly important for understanding the infectious disease transmission dynamics. In this study, we compare the transmission potentials of North American influenza A(H1N1)pdm09 derived from sequence data to that derived from surveillance data. The impact of the choice of tree-priors, informative epidemiological priors, and evolutionary parameters on the transmission potential estimation is evaluated. North American Influenza A(H1N1)pdm09 hemagglutinin (HA) gene sequences are analyzed using the coalescent and birth-death tree prior models to estimate the basic reproduction number (R 0 ). Epidemiological priors gathered from published literature are used to simulate the birth-death skyline models. Path-sampling marginal likelihood estimation is conducted to assess model fit. A bibliographic search to gather surveillance-based R 0 values were consistently lower (mean ≤ 1.2) when estimated by coalescent models than by the birth-death models with informative priors on the duration of infectiousness (mean ≥ 1.3 to ≤2.88 days). The user-defined informative priors for use in the birth-death model shift the directionality of epidemiological and evolutionary parameters compared to non-informative estimates. While there was no certain impact of clock rate and tree height on the R 0 estimation, an opposite relationship was observed between coalescent and birth-death tree priors. There was no significant difference (p = 0.46) between the birth-death model and surveillance R 0 estimates. This study concludes that tree-prior methodological differences may have a substantial impact on the transmission potential estimation as well as the evolutionary parameters. The study also reports a consensus between the sequence-based R 0 estimation and surveillance-based R 0 estimates. Altogether, these outcomes shed light on the potential role of phylodynamic modeling to augment existing surveillance and epidemiological activities to better assess and respond to emerging infectious diseases.

Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia.

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Methodological synthesis of Bayesian phylodynamics, HIV-TRACE, and GEE: HIV-1 transmission epidemiology in a racially/ethnically diverse Southern U.S. context.

This study introduces an innovative methodological approach to identify potential drivers of structuring HIV-1 transmission clustering patterns between different subpopulations in the culturally and racially/ethnically diverse context of Houston, TX, the largest city in the Southern United States. Using 6332 HIV-1 pol sequences from persons newly diagnosed with HIV during the period 2010-2018, we reconstructed HIV-1 transmission clusters, using the HIV-TRAnsmission Cluster Engine (HIV-TRACE); inferred demographic and risk parameters on HIV-1 transmission dynamics by jointly estimating viral transmission rates across racial/ethnic, age, and transmission risk groups; and modeled the degree of network connectivity by using generalized estimating equations (GEE). Our results indicate that Hispanics/Latinos are most vulnerable to the structure of transmission clusters and serve as a bridge population, acting as recipients of transmissions from Whites (3.0 state changes/year) and from Blacks (2.6 state changes/year) as well as sources of transmissions to Whites (1.8 state changes/year) and to Blacks (1.2 state changes/year). There were high rates of transmission and high network connectivity between younger and older Hispanics/Latinos as well as between younger and older Blacks. Prevention and intervention efforts are needed for transmission clusters that involve younger racial/ethnic minorities, in particular Hispanic/Latino youth, to reduce onward transmission of HIV in Houston.

Insecticide resistance in Anopheles stephensi in Somali Region, eastern Ethiopia.

BACKGROUND: The movement of malaria vectors into new areas is a growing concern in the efforts to control malaria. The recent report of Anopheles stephensi in eastern Ethiopia has raised the necessity to understand the insecticide resistance status of the vector in the region to better inform vector-based interventions. The aim of this study was to evaluate insecticide resistance in An. stephensi in eastern Ethiopia using two approaches: (1) World Health Organization (WHO) bioassay tests in An. stephensi; and (2) genetic analysis of insecticide resistance genes in An. stephensi in eastern Ethiopia. METHODS: Mosquito larvae and pupae were collected from Kebri Dehar. Insecticide susceptibility of An. stephensi was tested with malathion 5%, bendiocarb 0.1%, propoxur 0.1%, deltamethrin 0.05%, permethrin 0.75%, pirimiphos-methyl 0.25% and DDT 4%, according to WHO standard protocols. In this study, the knockdown resistance locus (kdr) in the voltage gated sodium channel (vgsc) and ace1R locus in the acetylcholinesterase gene (ace-1) were analysed in An. stephensi. RESULTS: All An. stephensi samples were resistant to carbamates, with mortality rates of 23% and 21% for bendiocarb and propoxur, respectively. Adult An. stephensi was also resistant to pyrethroid insecticides with mortality rates 67% for deltamethrin and 53% for permethrin. Resistance to DDT and malathion was detected in An. stephensi with mortality rates of 32% as well as An. stephensi was resistance to pirimiphos-methyl with mortality rates 14%. Analysis of the insecticide resistance loci revealed the absence of kdr L1014F and L1014S mutations and the ace1R G119S mutation. CONCLUSION: Overall, these findings support that An. stephensi is resistant to several classes of insecticides, most notably pyrethroids. However, the absence of the kdr L1014 gene may suggest non-target site resistance mechanisms. Continuous insecticide resistance monitoring should be carried out in the region to confirm the documented resistance and exploring mechanisms conferring resistance in An. stephensi in Ethiopia.

Evolution of endemic and sylvatic lineages of dengue virus.

Recent disease outbreaks have raised awareness of tropical pathogens, especially mosquito-borne viruses. Dengue virus (DENV) is a widely studied mammalian pathogen transmitted by various species of mosquito in the genus Aedes, especially Aedes aegypti and Aedes albopictus. The prevailing view of the research community is that endemic viral lineages that cause epidemics of DENV in humans have emerged over time from sylvatic viral lineages, which persist in wild, non-human primates. These notions have been examined by researchers through phylogenetic analyses of the envelope gene (E) from the four serotypes of DENV (serotypes DENV-1 to DENV-4). In these previous reports, researchers used visual inspection of a phylogeny in order to assert that sylvatic lineages lead to endemic clades. In making this assertion, these researchers also reasserted the model of periodic sylvatic to endemic disease outbreaks. Since that study, there has been a significant increase in data both in terms of metadata (e.g., place and host of isolation) and genetic sequences of DENV. Here, we re-examine the model of sylvatic to endemic shifts in viral lineages through a phylogenetic tree search and character evolution study of metadata on the tree. We built a dataset of nucleotide sequences for 188 isolates of DENV that have metadata on sylvatic or endemic sampling along with three orthologous sequences from West Nile virus as the outgroup for the phylogenetic analysis. In contrast to previous research, we find that there are several shifts from endemic to sylvatic lineages as well as sylvatic to endemic lineages, indicating a much more dynamic model of evolution. We propose that a model that allows oscillation between sylvatic and endemic hosts better captures the dynamics of DENV transmission.

First detection of Anopheles stephensi Liston, 1901 (Diptera: culicidae) in Ethiopia using molecular and morphological approaches.

Malaria is a major public health concern in Ethiopia. With the increase in malaria cases in the Somali Region of Ethiopia, understanding the distribution and identifying the species of malaria vectors is vital to public health. Here we report the first detection of Anopheles stephensi in Ethiopia, a malaria vector typically found in the Middle East, the Indian subcontinent, and China, but recently found in Djibouti. An entomological investigation was conducted during November to December 2016 in Kebri Dehar town of the Ethiopian Somali Regional State as ancillary work for Anopheles spp. surveillance. Mosquito larvae were collected from water reservoirs. Larvae were reared in the laboratory to the adult stage and identified morphologically. PCR and sequencing of cytochrome oxidase 1 (COI) and internal transcribed spacer 2 (ITS2) loci were performed. Basic Local Alignment Search Tool (BLAST) was used to compare sample sequences to sequences in the NCBI nucleotide database for species identification. To further analyze the relationship between the specimen we collected in Kebri Dehar and other Anopheles samples available in Genbank, phylogenetic analysis was performed using a maximum likelihood approach. Molecular and morphological results confirm specimens were An. stephensi. The closest high scoring hit was for all specimens was for the An. stephensi sequence. Independent phylogenetic analyses of COI and ITS2 sequences revealed in both cases strong bootstrap (100) support for our sequence forming a clade with other An. stephensi sequences to the exclusion of any other species of Anopheles. In conclusion, Anopheles stephensi is present in Kebri Dehar town in Ethiopia. These findings highlight the need for additional research to examine the role of An. stephensi in malaria transmission in Ethiopia.

Glucose-6-Phosphate Dehydrogenase Deficiency Genetic Variants in Malaria Patients in Southwestern Ethiopia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked erythrocyte enzyme disorder with relevance to malaria treatment policy. Treatment with the antimalarial primaquine can result in hemolytic anemia in G6PD-deficient patients. With increased interest in primaquine use, it is important to identify G6PD variants in Ethiopia to inform malaria treatment policy. In the present study, mutations in the G6PD gene are identified in a sample of patients with malaria in Jimma town in southwest Ethiopia. Plasmodium species of infection were confirmed using polymerase chain reaction (PCR) and gel electrophoresis. PCR and Sanger sequencing were performed to observe a portion of the G6PD gene where the common G6PD mutations (A376G, G202A, and C563T) are found. Molecular analysis revealed that most of the samples were single Plasmodium vivax infections (83.7%). For G6PD genotyping, A376G was detected in 23.26% of individuals, whereas G202A and C563T were absent. Three other uncommon mutations were identified: rs782669677 (535G→A), rs370658483, (485 + 37 G→T), and a new mutation at chrX:154535443(C→T). Bioinformatic analysis of these mutations' potential functional impact suggests minimal effect on protein function. The discovery of both common and uncommon G6PD mutations contributes to the discussion on G6PD deficiency and appropriate primaquine treatment in Ethiopia.

Molecular evolution of Zika virus as it crossed the Pacific to the Americas.

Zika virus was previously considered to cause only a benign infection in humans. Studies of recent outbreaks of Zika virus in the Pacific, South America, Mexico and the Caribbean have associated the virus with severe neuropathology. Viral evolution may be one factor contributing to an apparent change in Zika disease as it spread from Southeast Asia across the Pacific to the Americas. To address this possibility, we have employed computational tools to compare the phylogeny, geography, immunology and RNA structure of Zika virus isolates from Africa, Asia, the Pacific and the Americas. In doing so, we compare and contrast methods and results for tree search and rooting of Zika virus phylogenies. In some phylogenetic analyses we find support for the hypothesis that there is a deep common ancestor between African and Asian clades (the "Asia/Africa" hypothesis). In other phylogenetic analyses, we find that Asian lineages are descendent from African lineages (the "out of Africa" hypothesis). In addition, we identify and evaluate key mutations in viral envelope protein coding and untranslated terminal RNA regions. We find stepwise mutations that have altered both immunological motif sets and regulatory sequence elements. Both of these sets of changes distinguish viruses found in Africa from those in the emergent Asia-Pacific-Americas lineage. These findings support the working hypothesis that mutations acquired by Zika virus in the Pacific and Americas contribute to changes in pathology. These results can inform experiments required to elucidate the role of viral genetic evolution in changes in neuropathology, including microcephaly and other neurological and skeletomuscular issues in infants, and Guillain-Barré syndrome in adults.

Zika Virus: Medical Countermeasure Development Challenges.

INTRODUCTION: Reports of high rates of primary microcephaly and Guillain-Barré syndrome associated with Zika virus infection in French Polynesia and Brazil have raised concerns that the virus circulating in these regions is a rapidly developing neuropathic, teratogenic, emerging infectious public health threat. There are no licensed medical countermeasures (vaccines, therapies or preventive drugs) available for Zika virus infection and disease. The Pan American Health Organization (PAHO) predicts that Zika virus will continue to spread and eventually reach all countries and territories in the Americas with endemic Aedes mosquitoes. This paper reviews the status of the Zika virus outbreak, including medical countermeasure options, with a focus on how the epidemiology, insect vectors, neuropathology, virology and immunology inform options and strategies available for medical countermeasure development and deployment. METHODS: Multiple information sources were employed to support the review. These included publically available literature, patents, official communications, English and Lusophone lay press. Online surveys were distributed to physicians in the US, Mexico and Argentina and responses analyzed. Computational epitope analysis as well as infectious disease outbreak modeling and forecasting were implemented. Field observations in Brazil were compiled and interviews conducted with public health officials.