SeV infection measured by bioluminescence imaging in living mice after treatment with Dexa and/or Cy.

<p>(A) Drug treatment regimen and timing of infection. Arrows denote days on which drug injections were performed or 7,000 PFU Sendai virus (SeV) was intranasally inoculated in 5 μL PBS. Bioluminescence was measured after i.p. injection of 150 mg/kg D-luciferin and imaging with a Xenogen machine. (B–D) Bioluminescence of the nasal cavity (B), trachea (C), and lungs (D) in mice inoculated 1 day before starting drug treatment. Data shown are averages of 2 independent experiments with 10 mice at each time point. (E–G) Bioluminescence of the nasal cavity (E), trachea (F), and lungs (G) in mice inoculated 4 days after starting drug treatment. The data shown are averages of 3 independent experiments with 15 mice at each time point. For panels B-G, all groups were inoculated with SeV and symbols denote the following treatment groups: PBS (black circles), Dexa (green diamonds), Cy (orange squares), and Dexa + Cy (blue triangles). Lighter symbols in panels B-D correspond to groups inoculated with SeV 1 day before drug treatment, and darker symbols in panels E-G correspond to groups inoculated with SeV 4 days after drug treatment started. Error bars represent standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started.</p

Viral load in the respiratory tracts of mice inoculated with SeV 4 days after treatment with Dexa and/or Cy.

<p>(A–C) Viral loads in the nasopharynx (A), trachea (B), and lungs (C). The data shown are averages of 3 animals at each time point. Bar colors for the groups are as follows: PBS (black), Dexa (green), Cy (orange), and Dexa + Cy (blue). Dashed lines represent the limit of detection. Error bars represent standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. Statistics: 2-Way ANOVA; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Effects of SeV-specific hyperimmune pooled serum and monoclonal antibodies on controlling SeV infection in immunosuppressed mice.

<p>(A) Drug treatment regimen with SeV-specific hyperimmune serum or mAbs. Arrows denote days on which serum or monoclonal antibodies were i.p. injected, drug injections were performed, and 7,000 PFU Sendai virus (SeV) was intranasally inoculated in 5 μL PBS. (B–D) Bioluminescence in the nasopharynx (B), trachea (C), and lungs (D) after infection with SeV and the following conditions: no immunosuppression and no hyperimmune serum (black circles), Dexa + Cy without hyperimmune serum (blue triangles), Dexa + Cy with hyperimmune serum (brown diamonds), and Dexa + Cy with nonspecific control serum (dark purple triangles). (E-G) Bioluminescence in the nasopharynx (E), trachea (F), and lungs (G) after infection with SeV and the following conditions: no immunosuppression and no monoclonal antibodies (black circles), Dexa + Cy without monoclonal antibodies (blue triangles), Dexa + Cy + M16 [anti-F protein monoclonal antibody] (orange circles), Dexa + Cy + S2 [anti-HN protein monoclonal antibody] (green diamonds), and Dexa + Cy + isotype control mouse IgG (small purple squares). The data shown are averages of 5 mice per group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started.</p

Non-invasive Imaging of Sendai Virus Infection in Pharmacologically Immunocompromised Mice: NK and T Cells, but not Neutrophils, Promote Viral Clearance after Therapy with Cyclophosphamide and Dexamethasone

<div><p>In immunocompromised patients, parainfluenza virus (PIV) infections have an increased potential to spread to the lower respiratory tract (LRT), resulting in increased morbidity and mortality. Understanding the immunologic defects that facilitate viral spread to the LRT will help in developing better management protocols. In this study, we immunosuppressed mice with dexamethasone and/or cyclophosphamide then monitored the spread of viral infection into the LRT by using a noninvasive bioluminescence imaging system and a reporter Sendai virus (murine PIV type 1). Our results show that immunosuppression led to delayed viral clearance and increased viral loads in the lungs. After cessation of cyclophosphamide treatment, viral clearance occurred before the generation of Sendai-specific antibody responses and coincided with rebounds in neutrophils, T lymphocytes, and natural killer (NK) cells. Neutrophil suppression using anti-Ly6G antibody had no effect on infection clearance, NK-cell suppression using anti-NK antibody delayed clearance, and T-cell suppression using anti-CD3 antibody resulted in no clearance (chronic infection). Therapeutic use of hematopoietic growth factors G-CSF and GM-CSF had no effect on clearance of infection. In contrast, treatment with Sendai virus—specific polysera or a monoclonal antibody limited viral spread into the lungs and accelerated clearance. Overall, noninvasive bioluminescence was shown to be a useful tool to study respiratory viral progression, revealing roles for NK and T cells, but not neutrophils, in Sendai virus clearance after treatment with dexamethasone and cyclophosphamide. Virus-specific antibodies appear to have therapeutic potential.</p></div

Histopathologic changes 13 d.a.d.s. (9 d.p.i.) in the respiratory tracts of mice inoculated with SeV 4 days after treatment with Dexa and /or Cy.

<p>Sections of the nasal cavity (A) and lungs (B) were stained with hematoxylin and eosin (H&E) (left panels), with a mAb to CD3 to show T-cell infiltration (middle panels), or with a mAb to SeV (right panels). Sections from Dexa + Cy-treated mice (bottom panels) were compared to sections from untreated controls (upper panels). The data are representative of the 4 different animals in each group.</p

Effect of drug treatment on peripheral lymphocyte populations.

<p>(A) B cell, (B) CD4+ T cell, (C) CD8+ T cell, and (D) NK cell percentages were determined for peripheral blood collected at the indicated time points. Dexa and Cy injections were performed as described previously, and 7,000 PFU SeV was intranasally inoculated in 5 μL PBS at 4 d.a.d.s. Groups include PBS (black bars) and Dexa + Cy (blue bars). The data shown are averages of 5 mice per group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. * <i>P</i> < 0.05, *** <i>P</i> < 0.001.</p

Immunological responses in immunosuppressed mice inoculated with SeV at 4 d.a.d.s.

<p>(A) Percent change in starting weight. (B) Peripheral blood neutrophil counts. (C) Peripheral blood lymphocyte counts. (D) Ratio of spleen weight to total body weight. For panels A-D, groups are shown as follows: PBS (black circles), Dexa (green diamonds), Cy (orange squares), and Dexa + Cy (blue triangles). (E) Splenic neutrophil counts. (F) Splenic lymphocyte counts. (G) BALF neutrophil counts. (H) BALF lymphocyte counts. (I and J) SeV-specific IgG levels in the BALF (I) and serum (J) of mice infected with SeV 4 days after treatment with Dexa and/or Cy. (K) Chemokines/cytokines in the BALF of SeV-infected mice at 12 d.p.i. (16 d.a.d.s). For panels E-K, bars are colored as follows: PBS (black), Dexa (green), Cy (orange), and Dexa + Cy (blue). The data shown are a representative of 2 independent experiments with 3 to 5 mice in each group. Error bars represent standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started. Statistics: 2-way ANOVA; *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Mice infected with SeV after treatment with Dexa + Cy are protected from a secondary lethal challenge.

<p>(A) Serum SeV-specific IgG levels measured 1 day before challenge. The data are representative of 2 independent experiments with 3 mice per group. (B–D) Bioluminescence in the nasopharynx (B), trachea (C), and lungs (D) after challenge with SeV. The data are averages of 2 independent experiments with 8 mice per group. Error bars represent standard deviation. Statistics: 2-Way ANOVA; ***<i>P</i> < 0.001.</p

Effect of anti-neutrophil antibody Ly6G on viral clearance.

<p>(A) Drug treatment regimen with anti-neutrophil antibody (anti-Ly6G). Arrows denote days on which anti-Ly6G was i.p. injected, drug injections were performed, and 7,000 PFU Sendai virus (SeV) was intranasally inoculated in 5 μL PBS. (B) Peripheral blood neutrophil counts. (C) Peripheral blood lymphocyte counts. (D) Percent change in starting weight. (E-G) Bioluminescence in the nasopharynx (E), trachea (F), and lungs (G). Groups include PBS (black circles), Dexa + Cy (blue triangles), and Dexa + Cy + anti-Ly6G (red circles). The data shown are averages of 5 mice from each group. In all graphs, error bars represent the standard deviation. d.p.i., days postinfection; d.a.d.s., days after drug started.</p

Source data for S1 Fig.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.</div