Crimean-Congo Hemorrhagic Fever Virus Diversity and Reassortment, Pakistan, 2017-2020.

Sporadic cases and outbreaks of Crimean-Congo hemorrhagic fever (CCHF) have been documented across Pakistan since 1976; however, data regarding the diversity of CCHF virus (CCHFV) in Pakistan is sparse. We whole-genome sequenced 36 CCHFV samples collected from persons infected in Pakistan during 2017-2020. Most CCHF cases were from Rawalpindi (n = 10), followed by Peshawar (n = 7) and Islamabad (n = 4). Phylogenetic analysis revealed the Asia-1 genotype was dominant, but 4 reassorted strains were identified. Strains with reassorted medium gene segments clustered with Asia-2 (n = 2) and Africa-2 (n = 1) genotypes; small segment reassortments clustered with the Asia-2 genotype (n = 2). Reassorted viruses showed close identity with isolates from India, Iran, and Tajikistan, suggesting potential crossborder movement of CCHFV. Improved and continuous human, tick, and animal surveillance is needed to define the diversity of circulating CCHFV strains in Pakistan and prevent transmission.

Detection of Hantavirus during the COVID-19 Pandemic, Arizona, USA, 2020.

We identified 2 fatal cases of persons infected with hantavirus in Arizona, USA, 2020; 1 person was co-infected with SARS-CoV-2. Delayed identification of the cause of death led to a public health investigation that lasted ≈9 months after their deaths, which complicated the identification of a vector or exposure.

Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer.

Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

Completed genome segments of Maciel, Lechiguanas, and Laguna Negra orthohantaviruses.

New World orthohantaviruses are rodent-borne tri-segmented viruses that cause hantavirus cardiopulmonary syndrome in humans in the Americas. Molecular diagnostics for orthohantaviruses can be improved with more sequence data. Reported here are completed genomes for Lechiguanas, Maciel, and Laguna Negra viruses.

Human Orthohantavirus disease prevalence and genotype distribution in the U.S., 2008-2020: a retrospective observational study.

Background: In the United States (U.S.), hantavirus pulmonary syndrome (HPS) and non-HPS hantavirus infection are nationally notifiable diseases. Criteria for identifying human cases are based on clinical symptoms (HPS or non-HPS) and acute diagnostic results (IgM+, rising IgG+ titers, RT-PCR+, or immunohistochemistry (IHC)+). Here we provide an overview of diagnostic testing and summarize human Hantavirus disease occurrence and genotype distribution in the U.S. from 2008 to 2020. Methods: Epidemiological data from the national hantavirus registry was merged with laboratory diagnostic testing results performed at the CDC. Residual hantavirus-positive specimens were sequenced, and the available epidemiological and genetic data sets were linked to conduct a genomic epidemiological study of hantavirus disease in the U.S. Findings: From 1993 to 2020, 833 human hantavirus cases have been identified, and from 2008 to 2020, 335 human cases have occurred. Among New World (NW) hantavirus cases detected at the CDC diagnostic laboratory (representing 29.2% of total cases), most (85.0%) were detected during acute disease, however, some convalescent cases were detected in states not traditionally associated with hantavirus infections (Connecticut, Missouri, New Jersey, Pennsylvania, Tennessee, and Vermont). From 1993 to 2020, 94.9% (745/785) of U.S. hantaviruses cases were detected west of the Mississippi with 45.7% (359/785) in the Four Corners region of the U.S. From 2008 to 2020, 67.7% of NW hantavirus cases were detected between the months of March and August. Sequencing of RT-PCR-positive cases demonstrates a geographic separation of Orthohantavirus sinnombreense species [Sin Nombre virus (SNV), New York virus, and Monongahela virus]; however, there is a large gap in viral sequence data from the Northwestern and Central U.S. Finally, these data indicate that commercial IgM assays are not concordant with CDC-developed assays, and that "concordant positive" (i.e., commercial IgM+ and CDC IgM+ results) specimens exhibit clinical characteristics of hantavirus disease. Interpretation: Hantaviral disease is broadly distributed in the contiguous U.S, viral variants are localised to specific geographic regions, and hantaviral disease infrequently detected in most Southeastern states. Discordant results between two diagnostic detection methods highlight the need for an improved standardised testing plan in the U.S. Hantavirus surveillance and detection will continue to improve with clearly defined, systematic reporting methods, as well as explicit guidelines for clinical characterization and diagnostic criteria. Funding: This work was funded by core funds provided to the Viral Special Pathogens Branch at CDC.

HantaNet: A New MicrobeTrace Application for Hantavirus Classification, Genomic Surveillance, Epidemiology and Outbreak Investigations.

Hantaviruses zoonotically infect humans worldwide with pathogenic consequences and are mainly spread by rodents that shed aerosolized virus particles in urine and feces. Bioinformatics methods for hantavirus diagnostics, genomic surveillance and epidemiology are currently lacking a comprehensive approach for data sharing, integration, visualization, analytics and reporting. With the possibility of hantavirus cases going undetected and spreading over international borders, a significant reporting delay can miss linked transmission events and impedes timely, targeted public health interventions. To overcome these challenges, we built HantaNet, a standalone visualization engine for hantavirus genomes that facilitates viral surveillance and classification for early outbreak detection and response. HantaNet is powered by MicrobeTrace, a browser-based multitool originally developed at the Centers for Disease Control and Prevention (CDC) to visualize HIV clusters and transmission networks. HantaNet integrates coding gene sequences and standardized metadata from hantavirus reference genomes into three separate gene modules for dashboard visualization of phylogenetic trees, viral strain clusters for classification, epidemiological networks and spatiotemporal analysis. We used 85 hantavirus reference datasets from GenBank to validate HantaNet as a classification and enhanced visualization tool, and as a public repository to download standardized sequence data and metadata for building analytic datasets. HantaNet is a model on how to deploy MicrobeTrace-specific tools to advance pathogen surveillance, epidemiology and public health globally.

Systemic macrophage depletion inhibits Helicobacter bilis-induced proinflammatory cytokine-mediated typhlocolitis and impairs bacterial colonization dynamics in a BALB/c Rag2-/- mouse model of inflammatory bowel disease.

Helicobacter bilis, an enterohepatic helicobacter, is associated with chronic hepatitis in aged immunocompetent inbred mice and inflammatory bowel disease (IBD) in immunodeficient mice. To evaluate the role of macrophages in H. bilis-induced IBD, Rag2(-/-) BALB/c or wild-type (WT) BALB/c mice were either sham dosed or infected with H. bilis Missouri strain under specific-pathogen-free conditions, followed by an intravenous injection of a 0.2-ml suspension of liposomes coated with either phosphate-buffered saline (control) or clodronate (a macrophage depleting drug) at 15 weeks postinfection (wpi). At 16 wpi, the ceca of H. bilis-infected Rag2(-/-) mice treated with control liposomes had significantly higher histopathological lesional scores (for cumulative typhlitis index, inflammation, edema, epithelial defects, and hyperplasia) and higher counts of F4/80(+) macrophages and MPO(+) neutrophils compared to H. bilis-infected Rag2(-/-) mice treated with clodronate liposomes. In addition, cecal quantitative PCR analyses revealed a significant suppression in the expression of macrophage-related cytokine genes, namely, Tnfa, Il-1ß, Il-10, Cxcl1, and iNos, in the clodronate-treated H. bilis-infected Rag2(-/-) mice compared to the H. bilis-infected Rag2(-/-) control mice. Finally, cecal quantitative PCR analyses also revealed a significant reduction in bacterial colonization in the clodronate-treated Rag2(-/-) mice. Taken together, our results suggest that macrophages are critical inflammatory cellular mediators for promoting H. bilis-induced typhlocolitis in mice.

Lack of commensal flora in Helicobacter pylori-infected INS-GAS mice reduces gastritis and delays intraepithelial neoplasia.

BACKGROUND & AIMS: Transgenic FVB/N insulin-gastrin (INS-GAS) mice have high circulating gastrin levels, and develop spontaneous atrophic gastritis and gastrointestinal intraepithelial neoplasia (GIN) with 80% prevalence 6 months after Helicobacter pylori infection. GIN is associated with gastric atrophy and achlorhydria, predisposing mice to nonhelicobacter microbiota overgrowth. We determined if germfree INS-GAS mice spontaneously develop GIN and if H pylori accelerates GIN in gnotobiotic INS-GAS mice. METHODS: We compared gastric lesions, levels of messenger RNA, serum inflammatory mediators, antibodies, and gastrin among germfree and H pylori-monoinfected INS-GAS mice. Microbiota composition of specific pathogen-free (SPF) INS-GAS mice was quantified by pyrosequencing. RESULTS: Germfree INS-GAS mice had mild hypergastrinemia but did not develop significant gastric lesions until 9 months old and did not develop GIN through 13 months. H pylori monoassociation caused progressive gastritis, epithelial defects, oxyntic atrophy, marked foveolar hyperplasia, dysplasia, and robust serum and tissue proinflammatory immune responses (particularly males) between 5 and 11 months postinfection (P<0.05, compared with germfree controls). Only 2 of 26 female, whereas 8 of 18 male, H pylori-infected INS-GAS mice developed low to high-grade GIN by 11 months postinfection. Stomachs of H pylori-infected SPF male mice had significant reductions in Bacteroidetes and significant increases in Firmicutes. CONCLUSIONS: Gastric lesions take 13 months longer to develop in germfree INS-GAS mice than male SPF INS-GAS mice. H pylori monoassociation accelerated gastritis and GIN but caused less severe gastric lesions and delayed onset of GIN compared with H pylori-infected INS-GAS mice with complex gastric microbiota. Changes in gastric microbiota composition might promote GIN in achlorhydric stomachs of SPF mice.

Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

Immune-cognitive system connectivity reduces bumblebee foraging success in complex multisensory floral environments.

Bumblebees are declining at alarming rate worldwide, posing a significant threat to the function and diversity of temperate ecosystems. These declines have been attributed, in part, to the direct effect of specific pathogens on bumblebee survival. However, pathogens may also have a negative impact on host populations indirectly through immune-induced cognitive deficits in infected individuals. To gain greater insight into mechanisms and potential conservation implications of such 'immune-brain crosstalk' in bumblebees, we non-pathogenetically activated humoral and cellular immune pathways in individuals and then tested for long-term reductions in cognitive performance and foraging proficiency. We show that chronic activation of humoral, but not a cellular, immune pathways and effectors in foragers significantly reduces their ability to flexibly and efficiently harvest resources in multi-sensory floral environments for at least 7 days post-treatment. Humoral defense responses thus have the potential to confer significant foraging costs to bumblebee foragers over timeframes that would negatively impact colony growth and reproductive output under natural conditions. Our findings indicate that fitness effects of immune-brain crosstalk should be considered before attributing wild bumblebee decline to a particular pathogen species.

One size does not fit all: Caste and sex differences in the response of bumblebees (Bombus impatiens) to chronic oral neonicotinoid exposure.

Neonicotinoid insecticides have been implicated in the rapid global decline of bumblebees over recent years, particularly in agricultural and urban areas. While there is much known about neonicotinoid toxicity effects at the colony stage of the bumblebee annual cycle, far less is known about such effects at other stages critical for the maintenance of wild populations. In the present work, individual-based feeding assays were used to show that chronic consumption of the widely used neonicotinoid clothianidin at a field-realistic average rate of 3.6 and 4.0 ng/g·bee/day reduces survival of queen and male bumblebees, respectively, within a 7-day period. In contrast, worker survival was unaffected at a similar consumption rate of 3.9 ng/g·bee/day. To test the hypothesis that males have a lower tolerance for oral clothianidin exposure than workers due to their haploid genetic status, RNAseq analysis was used to compare the transcriptomic responses of workers and males to chronic intake of clothianidin at a sub-lethal dose of 0.37ng/bee/day for 5 days. Surprisingly, clothianidin consumption only altered the expression of 19 putative detoxification genes in a sex-specific manner, with 11/19 genes showing increased expression in workers. Sub-lethal clothianidin exposure also altered the expression of 40 genes associated with other major biological functions, including locomotion, reproduction, and immunity. Collectively, these results suggest that chronic oral toxicity effects of neonicotinoids are greatest during mating and nest establishment phases of the bumblebee life cycle. Chronic oral toxicity testing on males and queens is therefore required in order to fully assess the impact of neonicotinoids on wild bumblebee populations.

Helicobacter pylori infection does not promote hepatocellular cancer in a transgenic mouse model of hepatitis C virus pathogenesis.

Helicobacter pylori (H. pylori) and hepatitis C virus (HCV) infect millions of people and can induce cancer. We investigated if H. pylori infection promoted HCV-associated liver cancer. Helicobacter-free C3B6F1 wild-type (WT) and C3B6F1-Tg(Alb1-HCVN)35Sml (HT) male and female mice were orally inoculated with H. pylori SS1 or sterile media. Mice were euthanized at ~12 mo postinoculation and samples were collected for analyses. There were no significant differences in hepatocellular tumor promotion between WT and HT mice; however, HT female mice developed significantly larger livers with more hepatic steatosis than WT female mice. H. pylori did not colonize the liver nor promote hepatocellular tumors in WT or HT mice. In the stomach, H. pylori induced more corpus lesions in WT and HT female mice than in WT and HT male mice, respectively. The increased corpus pathology in WT and HT female mice was associated with decreased gastric H. pylori colonization, increased gastric and hepatic interferon gamma expression, and increased serum Th1 immune responses against H. pylori. HT male mice appeared to be protected from H. pylori-induced corpus lesions. Furthermore, during gastric H. pylori infection, HT male mice were protected from gastric antral lesions and hepatic steatosis relative to WT male mice and these effects were associated with increased serum TNF-α. Our findings indicate that H. pylori is a gastric pathogen that does not promote hepatocellular cancer and suggest that the HCV transgene is associated with amelioration of specific liver and gastric lesions observed during concurrent H. pylori infection in mice.

Colitis and colon cancer in WASP-deficient mice require helicobacter species.

BACKGROUND: Wiskott-Aldrich syndrome protein-deficient patients and mice are immunodeficient and can develop inflammatory bowel disease. The intestinal microbiome is critical to the development of colitis in most animal models, in which Helicobacter spp. have been implicated in disease pathogenesis. We sought to determine the role of Helicobacter spp. in colitis development in Wiskott-Aldrich syndrome protein-deficient (WKO) mice. METHODS: Feces from WKO mice raised under specific pathogen-free conditions were evaluated for the presence of Helicobacter spp., after which a subset of mice were rederived in Helicobacter spp.-free conditions. Helicobacter spp.-free WKO animals were subsequently infected with Helicobacter bilis. RESULTS: Helicobacter spp. were detected in feces from WKO mice. After rederivation in Helicobacter spp.-free conditions, WKO mice did not develop spontaneous colitis but were susceptible to radiation-induced colitis. Moreover, a T-cell transfer model of colitis dependent on Wiskott-Aldrich syndrome protein-deficient innate immune cells also required Helicobacter spp. colonization. Helicobacter bilis infection of rederived WKO mice led to typhlitis and colitis. Most notably, several H. bilis-infected animals developed dysplasia with 10% demonstrating colon carcinoma, which was not observed in uninfected controls. CONCLUSIONS: Spontaneous and T-cell transfer, but not radiation-induced, colitis in WKO mice is dependent on the presence of Helicobacter spp. Furthermore, H. bilis infection is sufficient to induce typhlocolitis and colon cancer in Helicobacter spp.-free WKO mice. This animal model of a human immunodeficiency with chronic colitis and increased risk of colon cancer parallels what is seen in human colitis and implicates specific microbial constituents in promoting immune dysregulation in the intestinal mucosa.

Prevalence of murine Helicobacter spp. Infection is reduced by restocking research colonies with Helicobacter-free mice.

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.

Helicobacter marmotae and novel Helicobacter and Campylobacter species isolated from the livers and intestines of prairie dogs.

Prairie dogs (Cynomys ludovicianus) are used to study the aetiology and prevention of gallstones because of the similarities of prairie dog and human bile gallstone composition. Epidemiological and experimental studies have suggested a connection between infection with Helicobacter species and cholesterol cholelithiasis, cholecystis and gallbladder cancer. Ten of the 34 prairie dogs in this study had positive Helicobacter species identified by PCR using Helicobacter genus-specific primers. Ten of 34 prairie dogs had positive Campylobacter species identified in the intestine by PCR with Campylobacter genus-specific primers. Six Helicobacter sp. isolates and three Campylobacter sp. isolates were identified taxonomically by 16S rRNA gene analysis. The prairie dog helicobacters fell into three clusters adjacent to Helicobacter marmotae. On the basis of 16S rRNA gene sequence analysis, three strains in two adjacent clusters were included in the species H. marmotae. Three strains were only 97.1 % similar to the sequence of H. marmotae and can be considered a novel species with the provisional designation Helicobacter sp. Prairie Dog 3. The prairie dog campylobacters formed a single novel cluster and represent a novel Campylobacter sp. with the provisional designation Campylobacter sp. Prairie Dog. They branched with Campylobacter cuniculorum at 96.3 % similarity and had the greatest sequence similarity to Campylobacter helveticus at 97.1 % similarity. Whether H. marmotae or the novel Helicobacter sp. and Campylobacter sp. identified in prairie dogs play a role in cholesterol gallstones or hepatobiliary disease requires further studies.

Helicobacter hepaticus--induced liver tumor promotion is associated with increased serum bile acid and a persistent microbial-induced immune response.

Chronic microbial infection influences cancer progression, but the mechanisms that link them remain unclear. Constitutive androstane receptor (CAR) is a nuclear receptor that regulates enzymes involved in endobiotic and xenobiotic metabolism. CAR activation is a mechanism of xenobiotic tumor promotion; however, the effects of chronic microbial infection on tumor promotion have not been studied in the context of CAR function. Here, we report that CAR limits the effects of chronic infection-associated progression of liver cancer. CAR knockout (KO) and wild-type (WT) male mice were treated with or without the tumor initiator diethylnitrosamine (DEN) at 5 weeks of age and then orally inoculated with Helicobacter hepaticus (Hh) or sterile media at 8 weeks of age. At approximately 50 weeks postinoculation, mice were euthanized for histopathologic, microbiological, molecular, and metabolomic analyses. Hh infection induced comparable hepatitis in WT and KO mice with or without DEN that correlated with significant upregulation of Tnfα and toll receptor Tlr2. Notably, DEN-treated Hh-infected KO mice exhibited increased numbers of liver lobes with dysplasia and neoplasia and increased multiplicity of neoplasia, relative to similarly treated WT mice. Enhanced tumor promotion was associated with decreased hepatic expression of P450 enzymes Cyp2b10 and Cyp3a11, increased expression of Camp, and increased serum concentrations of chenodeoxycholic acid. Together, our findings suggest that liver tumor promotion is enhanced by an impaired metabolic detoxification of endobiotics and a persistent microbial-induced immune response.

17ß-estradiol and tamoxifen prevent gastric cancer by modulating leukocyte recruitment and oncogenic pathways in Helicobacter pylori-infected INS-GAS male mice.

Helicobacter pylori infection promotes male predominant gastric adenocarcinoma in humans. Estrogens reduce gastric cancer risk and previous studies showed that prophylactic 17ß-estradiol (E2) in INS-GAS mice decreases H. pylori-induced carcinogenesis. We examined the effect of E2 and tamoxifen (TAM) on H. pylori-induced gastric cancer in male and female INS-GAS mice. After confirming robust gastric pathology at 16 weeks postinfection (WPI), mice were implanted with E2, TAM, both E2 and TAM, or placebo pellets for 12 weeks. At 28 WPI, gastric histopathology, gene expression, and immune cell infiltration were evaluated and serum inflammatory cytokines measured. After treatment, no gastric cancer was observed in H. pylori-infected males receiving E2 and/or TAM, whereas 40% of infected untreated males developed gastric cancer. E2, TAM, and their combination significantly reduced gastric precancerous lesions in infected males compared with infected untreated males (P < 0.001, 0.01, and 0.01, respectively). However, TAM did not alter female pathology regardless of infection status. Differentially expressed genes from males treated with E2 or TAM (n = 363 and n = 144, Q < 0.05) associated highly with cancer and cellular movement, indicating overlapping pathways in the reduction of gastric lesions. E2 or TAM deregulated genes associated with metastasis (PLAUR and MMP10) and Wnt inhibition (FZD6 and SFRP2). Compared with controls, E2 decreased gastric mRNA (Q < 0.05) and serum levels (P < 0.05) of CXCL1, a neutrophil chemokine, leading to decreased neutrophil infiltration (P < 0.01). Prevention of H. pylori-induced gastric cancer by E2 and TAM may be mediated by estrogen signaling and is associated with decreased CXCL1, decreased neutrophil counts, and downregulation of oncogenic pathways.