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Acute respiratory virus infection (ARI) induces CD8+ T cells with diminished cytokine production and functional impairment. The role of cellular mediators of immune impairment, specifically CD4+ regulatory T cells (Tregs), is incompletely understood in ARI. Tregs are known suppressors of effector T cell function, but whether they are detrimental or beneficial in ARI remains controversial. We show in this paper that Treg depletion leads to increased CD8+ T cell function and lower virus titer in mice infected with human metapneumovirus. We further demonstrate that Tregs play a temporal role in the immune response to human metapneumovirus and influenza: Treg depletion before infection pathologically reduces virus-specific CD8+ T cell numbers and delays virus clearance, whereas depletion 2 d postinoculation enhances CD8+ T cell functionality without reducing virus-specific CD8+ T cell numbers. Mechanistically, Treg depletion during immune priming led to impaired dendritic cell and CD8+ T cell migration. Further, early Treg depletion was associated with immune skewing toward a type 2 phenotype characterized by increased type 2 innate lymphoid cells and TH2 CD4+ T cells, which was not observed when Treg depletion was delayed until after inoculation. These results indicate that the presence of Tregs at inoculation is critical for efficient priming of the CD8+ T cell response to ARI, whereas later in infection, Tregs are dispensable for virus clearance. [ABSTRACT FROM AUTHOR]

Background Influenza C virus (ICV) is associated with acute respiratory illness. Yet ICV remains under recognized, with most previous studies using only culture to identify cases. Objectives To develop a sensitive and specific real-time RT-PCR assay for ICV that allows for rapid and accurate detection in a clinical or research setting. Study design Multiple ICV sequences obtained from GenBank were analyzed, including 141 hemagglutinin-esterase (HE), 106 matrix (M), and 97 nucleoprotein (NP) sequences. Primers and probes were designed based on conserved regions. Multiple primer-probe sets were tested against multiple ICV strains. Results The ICV M and NP genes offered the most conserved sequence regions. Primers and probes based on newer sequence data offered enhanced detection of ICV, especially for low titer specimens. An NP-targeted assay yielded the best performance and was capable of detecting 10–100 RNA copies per reaction. The NP assay detected multiple clinical isolates of ICV collected in a field epidemiology study conducted in Peru. Conclusions We report a new real-time RT-PCR assay for ICV with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Introduction: Despite evidence describing the burden of invasive non-typhoidal salmonella (iNTS) disease in sub-Saharan Africa, iNTS is not recognized as a priority within global health policy institutions. Recently, Salmonella enterica serovar Typhimurium, sequence type (ST) 313, has been identified as the predominant cause of iNTS disease in multiple sub-Saharan African countries. Materials and Methods: We conducted multilocus sequence typing (MLST) to determine the prevalence of the ST313 genotype in a sample of blood isolates from ambulatory HIV-infected Mozambican adults with iNTS disease. Results: Of the 29 samples of NTS obtained and analyzed by MLST, all (29/29) were assigned the ST313 sequence type based on the set of allele types derived from each of the seven loci. For quality control, five randomly selected strains taken from the original cultures were confirmed as ST313, and the positive control strain SL3261 (taken from the original culture) was categorized as S. Typhimurium ST19. Conclusion: S. Typhimurium ST313 is an important example of a widely distributed pathogen that lacks a coordinated strategy for control. The highly vulnerable populations at risk for ST313 infection in Mozambique, and within the region, would benefit greatly from the development of new policy and on-the-ground capacity to support increased surveillance, prevention, and treatment initiatives. [ABSTRACT FROM AUTHOR]

Purpose: To examine the role of DNA repair and altered thymidine analogues in altering the response to radiation during thymidine deprivation. Methods and Materials: Mismatch repair–deficient and -proficient cell lines HEC59 and HC-2.4 were treated with fluorodeoxyuridine (FUdR), azidothymidine (AZT), and irradiation either alone or in combination, and outcomes of clonogenic survival and cell-cycle distributions were determined. Results: Survival outcomes for all treatments were similar for both cell lines, suggesting that hMSH2 does not significantly influence thymidine deprivation toxicity or radiosensitization. The chain-terminating thymidine analogue AZT increased the toxicity of FUdR and increased DNA fragmentation. The combination of FUdR and AZT afforded greater radiosensitization than either drug alone. Drug enhancement ratios, the degree of excess radiation-induced cell death in drug-treated cultures compared with radiation alone for HEC59, were 1.2, 1.4, and 1.8 for AZT, FUdR, and the combination, respectively. Enhancement ratios for HC-2.4 were 1.3, 1.5, and 1.8 for AZT, FUdR, and the combination, respectively. Conclusion: Azidothymidine, a chain-terminating thymidine analogue, can enhance the radiosensitizing affects of thymidine deprivation. Deoxyribonucleic acid strand breaks may play an important role in the mechanism of thymidine deprivation–induced radiosensitization. [Copyright &y& Elsevier]

Purpose: Increased cellular sensitivity to ionizing radiation due to thymidine depletion is the basis of radiosensitization with fluoropyrimidine and methotrexate. The mechanism responsible for cytotoxicity has not been fully elucidated but appears to involve both the introduction of uracil into, and its removal from, DNA. The role of base excision repair of uracil and oxidatively damaged bases in creating the increased radiosensitization during thymidine depletion is examined.Methods and Materials: Isogenic strains of S. cerevisiae differing only at loci involved in DNA repair functions were exposed to aminopterin and sulfanilamide to induce thymidine deprivation. Cultures were irradiated and survival determined by clonogenic survival assay.Results: Strains lacking uracil base excision repair (BER) activities demonstrated less radiosensitization than the parental strain. Mutant strains continued to show partial radiosensitization with aminopterin treatment. Mutants deficient in BER of both uracil and oxidatively damaged bases did not demonstrate radiosensitization. A recombination deficient rad52 mutant strain was markedly sensitive to radiation; addition of aminopterin increased radiosensitivity only slightly. Radiosensitization observed in rad52 mutants was also abolished by deletion of the APN1, NTG1, and NTG2 genes.Conclusion: These data suggest radiosensitization during thymidine depletion is the result of BER activities directed at both uracil and oxidatively damaged bases. [ABSTRACT FROM AUTHOR]

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control on college campuses is challenging given communal living and student social dynamics. Understanding SARS-CoV-2 transmission among college students is important for the development of optimal control strategies. Methods SARS-CoV-2 nasal swab samples were collected from University of Pittsburgh students for symptomatic testing and asymptomatic surveillance from August 2020 through April 2021 from 3 campuses. Whole-genome sequencing (WGS) was performed on 308 samples, and contact tracing information collected from students was used to identify transmission clusters. Results We identified 31 Pangolin lineages of SARS-CoV-2, the majority belonging to B.1.1.7 (Alpha) and B.1.2 lineages. Contact tracing identified 142 students (46%) clustering with each other; WGS identified 53 putative transmission clusters involving 216 students (70%). WGS identified transmissions that were missed by contact tracing. However, 84 cases (27%) could not be linked by either WGS or contact tracing. Clusters were most frequently linked to students residing in the same dormitory, off-campus roommates, friends, or athletic activities. Conclusions The majority of SARS-CoV-2–positive samples clustered by WGS, indicating significant transmission across campuses. The combination of WGS and contact tracing maximized the identification of SARS-CoV-2 transmission on campus. WGS can be used as a strategy to mitigate, and further prevent transmission among students. [ABSTRACT FROM AUTHOR]

Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments. [ABSTRACT FROM AUTHOR]

Among 4200 adults who presented with acute respiratory symptoms at a variety of medical practice settings (November 2006 through May 2012), only 13 (0.3%) nasal/throat swabs were positive for influenza C. Influenza C was rarely associated with medical care visits in adults. [ABSTRACT FROM AUTHOR]

Human metapneumovirus (HMPV) is a major cause of respiratory disease. The role of NK cells in protection against HMPV is unclear. We show that while HMPV-infected C57BL/6 mice had higher numbers of functional lung NK cells than mock-treated mice, comparing NK cell-depleted and control mice did not reveal differences in lung viral titers, histopathology, cytokine levels, or T cell numbers or function. These data indicate that NK cells are not required for host control of HMPV. [ABSTRACT FROM AUTHOR]

Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin aVßl-promoted HMPV infection. Here we show that both HMPV F-mediated bind-ing and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the ab-sence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellu-lar receptors in a manner that is independent of the viral Gprotein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins. [ABSTRACT FROM AUTHOR]

Abstract: Background: Human metapneumovirus (HMPV) is a paramyxovirus with multiple genetic lineages that is a leading cause of acute respiratory disease. Several RT-PCR assays have been described based on limited available sequence data. Objectives: To develop a broadly reactive real-time RT-PCR assay for HMPV that allows for a rapid, sensitive, and specific detection in a clinical or research setting. Study design: Three published assays for HMPV were modified based on analysis of multiple HMPV sequences obtained from GenBank. Original and modified assays were tested against prototype HMPV strains from each genetic sublineage, multiple isolates of HMPV from different years, a collection of clinical specimens, and commercial validation panels. Results: A number of potential sequence mismatches with diverse HMPV strains were identified. Modifications were made to oligonucleotides to improve annealing efficiency. Primers and probes based on newer sequence data offered enhanced detection of all subgroups, especially for low titer specimens. The new primers and probe detected multiple clinical isolates of HMPV collected over a twenty-year period. The modified assay improved detection of HMPV in a panel of clinical specimens, and correctly identified HMPV samples in two commercial validation sets. Conclusions: We report a modified real-time RT-PCR assay for HMPV that detects all genetic lineages with high sensitivity. [Copyright &y& Elsevier]

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development. [ABSTRACT FROM AUTHOR]

Introduction: The disease burden and risk factors for respiratory syncytial virus (RSV) and human metapneumovirus (MPV) infections among children living in remote, rural areas remain unclear. Materials and Methods: We conducted a prospective, household-based cohort study of children aged <3 years living in remote rural highland communities in San Marcos, Cajamarca, Peru. Acute respiratory illnesses (ARI), including lower respiratory tract infection (LRTI), were monitored through weekly household visits from March 2009 through September 2011. Nasal swabs collected during ARI/LRTI were tested for RSV, MPV, and other respiratory viruses using real-time RT-PCR. Incidence rates and rate ratios were calculated using mixed effects Poisson regression. Results: Among 892 enrolled children, incidence rates of RSV and MPV ARI were 30 and 17 episodes per 100 child-years, respectively. The proportions of RSV and MPV ARI that presented as LRTI were 12.5% and 8.9%, respectively. Clinic visits for ARI and hospitalizations were significantly more frequent (all p values <0.05) among children with RSV (clinic 41% and hospital 5.3%) and MPV ARI (38% and 3.5%) when compared with other viral infections (23% and 0.7%) and infections without virus detected (24% and 0.6%). In multivariable analysis, risk factors for RSV detection included younger age (RR 1.02, 95% CI: 1.00-1.03), the presence of a smoker in the house (RR 1.63, 95% CI: 1.12-2.38), residing at higher altitudes (RR 1.93, 95% CI: 1.25-3.00 for 2nd compared to 1st quartile residents; RR 1.98, 95% CI: 1.26-3.13 for 3rd compared to 1st quartile residents). Having an unemployed household head was significantly associated with MPV risk (RR 2.11, 95% CI: 1.12-4.01). Conclusion: In rural high altitude communities in Peru, childhood ARI due to RSV or MPV were common and associated with higher morbidity than ARI due to other viruses or with no viral detections. The risk factors identified in this study may be considered for interventional studies to control infections by these viruses among young children from developing countries. [ABSTRACT FROM AUTHOR]

Highlights: [•] Studies of respiratory viruses frequently use nasal swabs and viral transport medium (NS). [•] Pneumococcal colonization studies use nasopharyngeal swabs and STGG medium (NP). [•] We compared RT-PCR viral detections in paired NS and NP collected from the same child on the same date. [•] We observed very high agreement in viral detections, and similar CT values in both strategies. [•] NP samples preserved in STGG allow efficient study of both pneumococcus and respiratory viruses. [Copyright &y& Elsevier]

Background: The Respiratory Infections in Andean Peruvian Children (RESPIRA-PERU) study enrolled children who participated in a community-cluster randomized trial of improved stoves, solar water disinfection, and kitchen sinks (IHIP trial) and children from additional Andean households. We quantified the burden of influenza-associated acute respiratory illness (ARI) in this household-based cohort. Methods: From May 2009 to September 2011, we conducted active weekly ARI surveillance in 892 children age <3 years, of whom 272 (30.5%) had participated in the IHIP trial. We collected nasal swabs during ARI, tested for influenza and other respiratory viruses by RT-PCR, and determined influenza incidence and risk factors using mixed-effects regression models. Results: The overall incidence of influenza-associated ARI was 36.6/100 child-years; incidence of influenza A, B, and C was 20.5, 8.7, and 5.2/100 child-years, respectively. Influenza C was associated with fewer days of subjective fever (median 1 vs. 2) and malaise (median 0 vs. 2) compared to influenza A. Non-influenza ARI also resulted in fewer days of fever and malaise, and fewer healthcare visits than influenza A-associated ARI. Influenza incidence varied by calendar year (80% occurred in the 2010 season) and IHIP trial participation. Among households that participated in the IHIP trial, influenza-associated ARI incidence was significantly lower in intervention than in control households (RR 0.40, 95% CI: 0.20–0.82). Conclusions: Influenza burden is high among Andean children. ARI associated with influenza A and B had longer symptom duration and higher healthcare utilization than influenza C-associated ARI or non-influenza ARI. Environmental community interventions may reduce influenza morbidity. [ABSTRACT FROM AUTHOR]

Viruses are leading causes of severe acute lower respiratory infections (LRIs). These infections evoke incomplete immunity, as individuals can be repeatedly reinfected throughout life. We report that acute viral LRI causes rapid pulmonary CD8+ cytotoxic T lymphocyte (TCD8) functional impairment via programmed death-1/programmed death ligand-1 (PD-1/PD-L1) signaling, a pathway previously associated with prolonged antigenic stimulation during chronic infections and cancer. PD-1-mediated TCD8 impairment occurred acutely in mice following infection with human metapneumovirus or influenza virus. Viral antigen was sufficient for PD-1 upregulation, but induction of PD-L1 was required for impairment. During secondary viral infection or epitope-only challenge, memory TCD8 rapidly reexpressed PD-1 and exhibited severe functional impairment. Inhibition of PD-1 signaling using monoclonal antibody blockade prevented TCD8 impairment, reduced viral titers during primary infection, and enhanced protection of immunized mice against challenge infection. Additionally, PD-1 and PD-L1 were upregulated in the lungs of patients with 2009 H1N1 influenza virus, respiratory syncytial virus, or parainfluenza virus infection. These results indicate that PD-1 mediates TCD8 functional impairment during acute viral infection and may contribute to recurrent viral LRIs. Therefore, the PD-1/PD-L1 pathway may represent a therapeutic target in the treatment of respiratory viruses. [ABSTRACT FROM AUTHOR]

Viruses are leading causes of severe acute lower respiratory infections (LRIs). These infections evoke incomplete immunity, as individuals can be repeatedly reinfected throughout life. We report that acute viral LRI causes rapid pulmonary CD8+ cytotoxic T lymphocyte (TCD8) functional impairment via programmed death-1/ programmed death ligand-1 (PD-1/PD-L1) signaling, a pathway previously associated with prolonged antigenic stimulation during chronic infections and cancer. PD-1-mediated TCD8 impairment occurred acutely in mice following infection with human metapneumovirus or influenza virus. Viral antigen was sufficient for PD-1 upregulation, but induction of PD-L1 was required for impairment. During secondary viral infection or epitope-only challenge, memory TCD8 rapidly reexpressed PD-1 and exhibited severe functional impairment. Inhibition of PD-1 signaling using monoclonal antibody blockade prevented TCD8 impairment, reduced viral titers during primary infection, and enhanced protection of immunized mice against challenge infection. Additionally, PD-1 and PD-L1 were upregulated in the lungs of patients with 2009 H1N1 influenza virus, respiratory syncytial virus, or parainfluenza virus infection. These results indicate that PD-1 mediates TCD8 functional impairment during acute viral infection and may contribute to recurrent viral LRIs. Therefore, the PD-1/PD-L1 pathway may represent a therapeutic target in the treatment of respiratory viruses. [ABSTRACT FROM AUTHOR]