Case of Human Orthohantavirus Infection, Michigan, USA, 2021.

Orthohantaviruses cause hantavirus cardiopulmonary syndrome; most cases occur in the southwest region of the United States. We discuss a clinical case of orthohantavirus infection in a 65-year-old woman in Michigan and the phylogeographic link of partial viral fragments from the patient and rodents captured near the presumed site of infection.

Choclo virus (CHOV) recovered from deep metatranscriptomics of archived frozen tissues in natural history biorepositories.

BACKGROUND: Hantaviruses are negative-stranded RNA viruses that can sometimes cause severe disease in humans; however, they are maintained in mammalian host populations without causing harm. In Panama, sigmodontine rodents serve as hosts to transmissible hantaviruses. Due to natural and anthropogenic forces, these rodent populations are having increased contact with humans. METHODS: We extracted RNA and performed Illumina deep metatranscriptomic sequencing on Orthohantavirus seropositive museum tissues from rodents. We acquired sequence reads mapping to Choclo virus (CHOV, Orthohantavirus chocloense) from heart and kidney tissue of a two-decade old frozen museum sample from a Costa Rican pygmy rice rat (Oligoryzomys costaricensis) collected in Panama. Reads mapped to the CHOV reference were assembled and then validated by visualization of the mapped reads against the assembly. RESULTS: We recovered a 91% complete consensus sequence from a reference-guided assembly to CHOV with an average of 16X coverage. The S and M segments used in our phylogenetic analyses were nearly complete (98% and 99%, respectively). There were 1,199 ambiguous base calls of which 93% were present in the L segment. Our assembled genome varied 1.1% from the CHOV reference sequence resulting in eight nonsynonymous mutations. Further analysis of all publicly available partial S segment sequences support a clear relationship between CHOV clinical cases and O. costaricensis acquired strains. CONCLUSIONS: Viruses occurring at extremely low abundances can be recovered from deep metatranscriptomics of archival tissues housed in research natural history museum biorepositories. Our efforts resulted in the second CHOV genome publicly available. This genomic data is important for future surveillance and diagnostic tools as well as understanding the evolution and pathogenicity of CHOV.

Distribution and prevalence of Sin Nombre hantavirus in rodent species in eastern New Mexico.

Orthohantaviruses are diverse zoonotic RNA viruses. Small mammals, such as mice and rats are common chronic, asymptomatic hosts that transmit the virus through their feces and urine. In North America, hantavirus infection primarily causes hantavirus cardiopulmonary syndrome (HCPS), which has a mortality rate of nearly 36%. In the United States of America, New Mexico (NM) is leading the nation in the number of HCPS-reported cases (N = 129). However, no reported cases of HCPS have occurred within eastern NM. In this study, we assessed the prevalence of Sin Nombre virus (SNV) in rodent assemblages across eastern NM, using RT-qPCR. We screened for potential rodent hosts in the region, as well as identified areas that may pose significant infection risk to humans. We captured and collected blood and lung tissues from 738 rodents belonging to 23 species. 167 individuals from 16 different species were positive for SNV RNA by RT-qPCR, including 6 species unreported in the literature: Onychomys leucogaster (Northern grasshopper mouse), Dipodomys merriami (Merriam's kangaroo rat), Dipodomys ordii (Ord's kangaroo rat), Dipodomys spectabilis (Banner-tailed kangaroo rat), Perognathus flavus (Silky pocket mouse), and Chaetodipus hispidus (Hispid pocket mouse). The infection rates did not differ between sexes or rodent families (i.e., Cricetidae vs. Heteromyidae). Generalized linear model showed that disturbed habitat types positively influenced the prevalence of SNV at sites of survey. Overall, the results of this study indicate that many rodent species in east New Mexico have the potential to maintain SNV in the environment, but further research is needed to assess species specific infectivity mechanisms and potential risk to humans.

G-CSF Is a Novel Mediator of T-Cell Suppression and an Immunotherapeutic Target for Women with Colon Cancer.

PURPOSE: G-CSF enhances colon cancer development. This study defines the prevalence and effects of increased G-CSF signaling in human colon cancers and investigates G-CSF inhibition as an immunotherapeutic strategy against metastatic colon cancer. EXPERIMENTAL DESIGN: Patient samples were used to evaluate G-CSF and G-CSF receptor (G-CSFR) levels by IHC with sera used to measure G-CSF levels. Peripheral blood mononuclear cells were used to assess the rate of G-CSFR+ T cells and IFNγ responses to chronic ex vivo G-CSF. An immunocompetent mouse model of peritoneal metastasis (MC38 cells in C57Bl/6J) was used to determine the effects of G-CSF inhibition (αG-CSF) on survival and the tumor microenvironment (TME) with flow and mass cytometry. RESULTS: In human colon cancer samples, the levels of G-CSF and G-CSFR are higher compared to normal colon tissues from the same patient. High patient serum G-CSF is associated with increases in markers of poor prognosis, (e.g., VEGF, IL6). Circulating T cells from patients express G-CSFR at double the rate of T cells from controls. Prolonged G-CSF exposure decreases T cell IFNγ production. Treatment with αG-CSF shifts both the adaptive and innate compartments of the TME and increases survival (HR, 0.46; P = 0.0237) and tumor T-cell infiltration, activity, and IFNγ response with greater effects in female mice. There is a negative correlation between serum G-CSF levels and tumor-infiltrating T cells in patient samples from women. CONCLUSIONS: These findings support G-CSF as an immunotherapeutic target against colon cancer with greater potential benefit in women.

An analysis of sexual dimorphism in the tumor microenvironment of colorectal cancer.

Women with colorectal cancer (CRC) have survival advantages over men, yet the underlying mechanisms are unclear. T cell infiltration within the CRC tumor microenvironment (TME) correlates strongly with survival. We hypothesized that women with CRC have increased T cell infiltration and differential gene expression in the TME compared to men. Tissue microarrays comprising primary tumor, tumor infiltrated lymph nodes, and uninvolved colon were created from CRC patients. Proportions of CD4 positive (CD4+) and CD8 positive (CD8+) T cells were identified using immunohistochemistry. TME immune- and cancer-related genetic expression from primary and metastatic CRC tumor were also evaluated via the NanoStringIO360 panel and The Cancer Genome Atlas Project database. CD4+ was higher in tumor samples from women compared to men (22.04% vs. 10.26%, p=0.002) and also in lymph node samples (39.54% vs. 8.56%, p=0.001). CD8+ was increased in uninvolved colon from women compared to men (59.40% vs. 43.61%, p=0.015), and in stage I/II tumors compared to III/IV in all patients (37.01% vs. 23.91%, p=0.009). Top CD8+ tertile patients survived longer compared to the bottom (43.9 months vs. 25.3 months, p=0.007). Differential gene expression was observed in pathways related to Treg function, T cell activity, and T cell exhaustion, amongst several others, in women compared to men. Thus, significant sexual dimorphism exists in the TME that could contribute to survival advantages observed in female patients with CRC.

Use of a Novel Detection Tool to Survey Orthohantaviruses in Wild-Caught Rodent Populations.

Orthohantaviruses are negative-stranded RNA viruses with trisegmented genomes that can cause severe disease in humans and are carried by several host reservoirs throughout the world. Old World orthohantaviruses are primarily located throughout Europe and Asia, causing hemorrhagic fever with renal syndrome, and New World orthohantaviruses are found in North, Central, and South America, causing hantavirus cardiopulmonary syndrome (HCPS). In the United States, Sin Nombre orthohantavirus (SNV) is the primary cause of HCPS with a fatality rate of ~36%. The primary SNV host reservoir is thought to be the North American deer mouse, Peromyscus maniculatus. However, it has been shown that other species of Peromyscus can carry different orthohantaviruses. Few studies have systemically surveyed which orthohantaviruses may exist in wild-caught rodents or monitored spillover events into additional rodent reservoirs. A method for the rapid detection of orthohantaviruses is needed to screen large collections of rodent samples. Here, we report a pan-orthohantavirus, two-step reverse-transcription quantitative real-time PCR (RT-qPCR) tool designed to detect both Old and New World pathogenic orthohantavirus sequences of the S segment of the genome and validated them using plasmids and authentic viruses. We then performed a screening of wild-caught rodents and identified orthohantaviruses in lung tissue, and we confirmed the findings by Sanger sequencing. Furthermore, we identified new rodent reservoirs that have not been previously reported as orthohantavirus carriers. This novel tool can be used for the efficient and rapid detection of various orthohantaviruses, while uncovering potential new orthohantaviruses and host reservoirs that may otherwise go undetected.

Tracing Transmission of Sin Nombre Virus and Discovery of Infection in Multiple Rodent Species.

Sin Nombre orthohantavirus (SNV), a negative-sense, single-stranded RNA virus that is carried and transmitted by the North American deer mouse Peromyscus maniculatus, can cause infection in humans through inhalation of aerosolized excreta from infected rodents. This infection can lead to hantavirus cardiopulmonary syndrome (HCPS), which has an ∼36% case-fatality rate. We used reverse transcriptase quantitative PCR (RT-qPCR) to confirm SNV infection in a patient and identified SNV in lung tissues in wild-caught rodents from potential sites of exposure. Using viral whole-genome sequencing (WGS), we identified the likely site of transmission and discovered SNV in multiple rodent species not previously known to carry the virus. Here, we report, for the first time, the use of SNV WGS to pinpoint a likely site of human infection and identify SNV simultaneously in multiple rodent species in an area of known host-to-human transmission. These results will impact epidemiology and infection control for hantaviruses by tracing zoonotic transmission and investigating possible novel host reservoirs. IMPORTANCE Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nombre orthohantavirus (SNV) is the main cause of hantavirus disease in North America. In this study, we sequenced SNV from an infected patient and wild-caught rodents to trace the location of infection. We also discovered SNV in rodent species not previously known to carry SNV. These studies demonstrate for the first time the use of virus sequencing to trace the transmission of SNV and describe infection in novel rodent species.

COVID-19 global pandemic planning: Dry heat incubation and ambient temperature fail to consistently inactivate SARS-CoV-2 on N95 respirators.

The ongoing pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has placed a substantial strain on the supply of personal protective equipment, particularly the availability of N95 respirators for frontline healthcare personnel. These shortages have led to the creation of protocols to disinfect and reuse potentially contaminated personal protective equipment. A simple and inexpensive decontamination procedure that does not rely on the use of consumable supplies is dry heat incubation. Although reprocessing with this method has been shown to maintain the integrity of N95 respirators after multiple decontamination procedures, information on the ability of dry heat incubation to inactivate SARS-CoV-2 is largely unreported. Here, we show that dry heat incubation does not consistently inactivate SARS-CoV-2-contaminated N95 respirators, and that variation in experimental conditions can dramatically affect viability of the virus. Furthermore, we show that SARS-CoV-2 can survive on N95 respirators that remain at room temperature for at least five days. Collectively, our findings demonstrate that dry heat incubation procedures and ambient temperature for five days are not viable methods for inactivating SARS-CoV-2 on N95 respirators for potential reuse. We recommend that decontamination procedures being considered for the reuse of N95 respirators be validated at each individual site and that validation of the process must be thoroughly conducted using a defined protocol.

The iminosugars celgosivir, castanospermine and UV-4 inhibit SARS-CoV-2 replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented challenge for health care and the global economy. Repurposing drugs that have shown promise in inhibiting other viral infections could allow for more rapid dispensation of urgently needed therapeutics. The Spike protein of SARS-CoV-2 is extensively glycosylated with 22 occupied N glycan sites and is required for viral entry. In other glycosylated viral proteins, glycosylation is required for interaction with calnexin and chaperone-mediated folding in the endoplasmic reticulum, and prevention of this interaction leads to unfolded viral proteins and thus inhibits viral replication. As such, we investigated two iminosugars, celgosivir, a prodrug of castanospermine, and UV-4, or N-(9-methoxynonyl)-1-deoxynojirimycin, a deoxynojirimycin derivative. Iminosugars are known inhibitors of the α-glucosidase I and II enzymes and were effective at inhibiting authentic SARS-CoV-2 viral replication in a cell culture system. Celgosivir prevented SARS-CoV-2-induced cell death and reduced viral replication and Spike protein levels in a dose-dependent manner in culture with Vero E6 cells. Castanospermine, the active form of celgosivir, was also able to inhibit SARS-CoV-2, confirming the canonical castanospermine mechanism of action of celgosivir. The monocyclic UV-4 also prevented SARS-CoV-2-induced death and reduced viral replication after 24 h of treatment, although the reduction in viral copies was lost after 48 h. Our findings suggest that iminosugars should be urgently investigated as potential SARS-CoV-2 inhibitors.

The role of sex in the innate and adaptive immune environment of metastatic colorectal cancer.

BACKGROUND: Women with colorectal cancer (CRC) have a significant survival advantage over men. Sex influences on the tumour microenvironment (TME) are not well characterised, despite the importance of immune response in CRC. We hypothesised that sex-divergent immune responses could contribute to survival. METHODS: Using a murine model of metastatic CRC, we examined T cells, macrophages, and cytokines locally and systemically. TME and serum cytokines were measured by multiplex bead-based arrays, while FCA was used to identify cells and phenotypes. IHC provided spatial confirmation of T cell infiltration. RESULTS: Females had increased survival and T cell infiltration. CD8, CD4 and Th2 populations correlated with longer survival. Males had increased serum levels of chemokines and inflammation-associated cytokines. Within the TME, males had lower cytokine levels than females, and a shallower cytokine gradient to the periphery. Female tumours had elevated IL-10+ macrophages, which correlated with survival. CONCLUSIONS: These data demonstrate survival-associated differences in the immune response of males and females to metastatic CRC. Females showed changes in cytokine production accompanied by increased immune cell populations, biased toward Th2-axis phenotypes. Key differences in the immune response to CRC correlated with survival in this model. These differences support a multi-faceted shift across the TME.

High levels of tumor-associated neutrophils are associated with improved overall survival in patients with stage II colorectal cancer.

Conflicting reports regarding whether high tumor-associated neutrophils (TAN) are associated with outcomes in colorectal cancer (CRC) exist. Previous investigators have counted TAN using non-neutrophil-specific immunohistochemistry (IHC) stains. We examined whether TAN levels as determined by multi-field manual counting would predict prognosis. IRB approval was obtained and two pathologists, blinded to stage/outcome, counted TAN in 20 high power fields (HPF) per specimen. TAN score was defined as the mean of these counts. High TAN was defined as at or greater than the median score for that stage. Demographics, tumor characteristics, and overall survival (OS) were obtained from the records and examined for association with TAN score. IHC for arginase expression was performed in a subset of samples. 221 patients were included. Stage II patients with high TAN scores had an OS of 232 months as compared to those with low TAN (OS = 85 months, p = 0.03). The survival benefit persisted in multivariable analysis (HR 0.48, CI 0.25-0.91, p = 0.026) controlling for age and sex. Women had increased survival as compared to men, and there were no significant prognostic associations with TAN count in stage III/IV patients, although there were only 12 stage IV patients. Arginase staining did not provide additional information. Stage II colorectal cancer patients with high TAN live nearly 3 times longer than those with low TAN. Women with stage II disease and high TAN counts appear to be driving the survival benefit seen in the stage II patients and have increased overall survival in all stages.

Blockade of MK2 is protective in inflammation-associated colorectal cancer development.

Chronic inflammation is a risk factor for colorectal cancer. The MAPK-activated protein kinase 2 (MK2) pathway controls multiple cellular processes including p38-dependent inflammation. This is the first study to investigate the role of MK2 in development of colitis-associated colon cancer (CAC). Herein, we demonstrate that MK2(-/-) mice are highly resistant to neoplasm development when exposed to AOM/DSS, while wild type (WT) C57BL/6 develop multiple neoplasms with the same treatment. MK2-specific cytokines IL-1, IL-6 and TNF-α were substantially decreased in AOM/DSS treated MK2(-/-) mouse colon tissues compared with WT mice, which coincided with a marked decrease in macrophage influx. Restoring MK2-competent macrophages by injecting WT bone marrow derived macrophages into MK2(-/-) mice led to partial restoration of inflammatory cytokine production with AOM/DSS treatment; however, macrophages were not sufficient to induce neoplasm development. These results indicate that MK2 functions as an inflammatory regulator to promote colonic neoplasm development and may be a potential target for CAC.

Anti-G-CSF treatment induces protective tumor immunity in mouse colon cancer by promoting protective NK cell, macrophage and T cell responses.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is highly expressed in human and mouse colorectal cancers (CRC). We previously reported that G-CSF stimulated human CRC cell growth and migration, therefore in this study we sought to examine the therapeutic potential of anti-G-CSF treatment for CRC. G-CSF is known to mobilize neutrophils, however its impact on other immune cells has not been well examined. Here, we investigated the effects of therapeutic anti-G-CSF treatment on CRC growth and anti-tumor immune responses. C57BL/6 mice treated with azoxymethane/dextran sodium sulfate (AOM/DSS) to induce neoplasms were administered anti-G-CSF or isotype control antibodies three times a week for three weeks. Animals treated with anti-G-CSF antibodies had a marked decrease in neoplasm number and size compared to the isotype control group. Colon neutrophil and macrophage frequency were unchanged, but the number of macrophages producing IL-10 were decreased while IL-12 producing macrophages were increased. NK cells were substantially increased in colons of anti-G-CSF treated mice, along with IFNγ producing CD4(+) and CD8(+) T cells. These studies are the first to indicate a crucial role for G-CSF inhibition in promoting protective anti-tumor immunity, and suggest that anti-G-CSF treatment is a potential therapeutic approach for CRC.

Chronic macrophage migration inhibitory factor exposure induces mesenchymal epithelial transition and promotes gastric and colon cancers.

Macrophage Migration Inhibitory Factor (MIF) is an inflammatory cytokine that is highly produced in gastrointestinal cancers. Since chronic inflammation is a risk factor for tumorigenesis in these cancers, in this study, the role of MIF in pro-tumorigenic events was examined. MIF and its receptor, CD74, were examined in gastric and colon tumors and found to be increased in most tumors with significantly higher expression in tumors from patients with lymph node metastasis. MIF was also found to be highly produced by cancer associated fibroblasts isolated from human tumors compared to fibroblasts from matched normal tissues from uninvolved areas. Fibroblast-produced MIF highly increased GI cancer cell proliferation, which was decreased upon neutralizing MIF or CD74. Chronic MIF treatment led to sustained proliferation and signaling events in non-transformed GI fibroblast cells, which was maintained upon removing MIF treatment for 8 weeks. Additionally, chronic treatment of normal GI cells expressing fibroblast markers for up to 16 weeks with MIF led to a drastic decrease of fibroblast markers with concurrent increase of epithelial markers. Transformation was examined by telomerase and focus forming assays. These results suggest the MIF promotes mesenchymal epithelial transition, cell transformation and tumorigenesis in GI cancers, and thus may be an important link between chronic inflammation and tumorigenesis.

Powassan virus in mammals, Alaska and New Mexico, U.S.A., and Russia, 2004-2007.

Powassan virus is endemic to the United States, Canada, and the Russian Far East. We report serologic evidence of circulation of this virus in Alaska, New Mexico, and Siberia. These data support further studies of viral ecology in rapidly changing Arctic environments.

Seroprevalence of Powassan virus in New England deer, 1979-2010.

Powassan virus and its subtype, deer tick virus, are closely related tick-borne flaviviruses that circulate in North America. The incidence of human infection by these agents appears to have increased in recent years. To define exposure patterns among white-tailed deer, potentially useful sentinels that are frequently parasitized by ticks, we screened serum samples collected during 1979-2010 in Connecticut, Maine, and Vermont for neutralizing antibody by using a novel recombinant deer tick virus-West Nile virus chimeric virus. Evidence of exposure was detected in all three states. Overall our results demonstrate that seroprevalence is variable in time and space, suggesting that risk of exposure to Powassan virus is similarly variable.

Stromal cells induce Th17 during Helicobacter pylori infection and in the gastric tumor microenvironment.

Gastric cancer is associated with chronic inflammation and Helicobacter pylori infection. Th17 cells are CD4(+) T cells associated with infections and inflammation; but their role and mechanism of induction during carcinogenesis is not understood. Gastric myofibroblasts/fibroblasts (GMF) are abundant class II MHC expressing cells that act as novel antigen presenting cells. Here we have demonstrated the accumulation of Th17 in H. pylori-infected human tissues and in the gastric tumor microenvironment. GMF isolated from human gastric cancer and H. pylori infected tissues co-cultured with CD4(+) T cells induced substantially higher levels of Th17 than GMF from normal tissues in an IL-6, TGF-ß, and IL-21 dependent manner. Th17 required interaction with class II MHC on GMF for activation and proliferation. These studies suggest that Th17 are induced during both H. pylori infection and gastric cancer in the inflammatory milieu of gastric stroma and may be an important link between inflammation and carcinogenesis.

Homogeneity of Powassan virus populations in naturally infected Ixodes scapularis.

Powassan virus (POWV, Flaviviridae: Flavivirus) is the sole North American member of the tick-borne encephalitis complex and consists of two distinct lineages that are maintained in ecologically discrete enzootic transmission cycles. The underlying genetic mechanisms that lead to niche partitioning in arboviruses are poorly understood. Therefore, intra- and interhost genetic diversity was analyzed to determine if POWV exists as a quasispecies in nature and quantify selective pressures within and between hosts. In contrast to previous reports for West Nile virus (WNV), significant intrahost genetic diversity was not observed. However, pN (0.238) and d(N)/d(S) ratios (0.092) for interhost diversity were similar to those of WNV. Combined, these data suggest that purifying selection and/or population bottlenecks constrain quasispecies diversity within ticks. These same selective and stochastic mechanisms appear to drive minor sequence changes between ticks. Moreover, Powassan virus populations seem not to be structured as quasispecies in naturally infected adult deer ticks.

Persistent neuropsychological impairment associated with West Nile virus infection.

West Nile virus infection can result in prolonged subjective complaints of cognitive and functional decline even in the absence of a neuroinvasive form of infection. Persistent cognitive and functional complaints could be a result of general somatic symptoms, emotional distress, or residual central nervous system damage or dysfunction. Most studies of cognition in postacute West Nile virus infection rely on self-report. This descriptive study aimed to document cognitive deficits in a sample of the 2003 infected population reported in New Mexico. Patients with clinically defined neuroinvasive disease or who were impaired on brief mental status screening were seen for comprehensive neuropsychological assessment. We found that one year after symptom onset, more than half of the sample had objectively measurable neuropsychological impairment in at least two cognitive domains. Impairment was not related to subjective complaints of physical or emotional distress, or premorbid intellectual abilities. Persistent cognitive impairment in West Nile virus infection may be due to prolonged or permanent damage to the central nervous system.

Potential renal sequelae in survivors of hantavirus cardiopulmonary syndrome.

Although other hantaviruses are associated with renal manifestations, hantavirus cardiopulmonary syndrome (HCPS) has not been associated with such sequelae. The HCPS survivors were prospectively evaluated for renal complications. Subjects underwent yearly evaluation, laboratory studies, and 24-hour urine collection. Thirty subjects were evaluated after recovery from HCPS with the first follow-up at a median of 7.4 months after discharge. Subjects were a wide age range (18-51) but had an equal gender composition. Eighteen of 30 (60%) returned for > 1 evaluation. Half (15/30) had a 24-hour urine collection with > 150 mg of total protein and 6 had > 300 mg. Seven had a Cockcroft-Gault creatinine clearance (CrClCG) < 90 mL/min/1.73 m2 and 2 were < 60. Fifty-three percent met the definition of chronic kidney disease. Those treated with extracorporeal membrane oxygenation had less renal sequelae (P = 0.035). Our data suggest that renal sequelae may occur in HCPS. Further study of renal complications of New World hantavirus infections are needed.