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Aerosol-generating medical procedures (AGMPs) are increasingly being recognized as important sources for nosocomial transmission of emerging viruses. Intubation was investigated as a possible cause of Ebola virus (EBOV) transmission among health-care workers (HCWs) in the United States. Additionally, the high rate of nosocomial transmission of Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) caused speculation about the role of AGMPs. Crimean–Congo hemorrhagic fever orthonairovirus (CCHFV), was also associated with nosocomial infection secondary to AGMPs. While guidelines were developed for performing AGMPs on patients with certain viral infections, assessing and understanding the risk that specific viruses and AGMPs pose for nosocomial transmission could improve infection control practices, as well as reveal relationships in virus transmission.
Despite the perceived importance of AGMPs in nosocomial transmission of viruses and other infectious agents, scarce empirical or quantitative evidence exists. In order to assess the risk that certain viruses and AGMPs create for nosocomial transmission, we first need to identify potential AGMPs and viruses. The second step is then to determine the risk associated with these viruses and procedures, either through retrospective analysis, investigating the circumstances of nosocomial transmission, or through experiments, such as using air sampling during AGMPs to determine the risk of generating infectious virus-laden aerosols. Lastly, we can use this knowledge to re-evaluate current guidelines and communicate which viruses and AGMPs pose the highest risk for nosocomial transmission.
Vaccinia virus (VV) is a member of the Poxviridae, which constitute a large family of enveloped DNA viruses and replicate entirely in the cytoplasm of the infected cells with a linear double-stranded DNA genome of 130–300 kilo base pairs. Poxviruses have a broad range of eukaryotic hosts including mammals, birds, reptiles and insects and can grow in many cell lines in vitro. Some poxviruses are causative agents of human diseases. Variola virus caused a deadly human disease smallpox until its global eradication in 1977, in which VV was used as a vaccine. Other poxviruses causing human diseases are molluscum contagiosum virus and the zoonotic monkeypox virus. Notably, variola and monkeypox viruses are transmitted to humans by respiratory route, whereas molluscum contagiosum virus is mainly transmitted through the skin. Variola and monkeypox viruses cause systemic infections with high levels of lethality, but the details of their pathogenesis are not well-understood.
Intranasal inoculation of different VV strains in mice shows different levels of virulence and only neurovirulent strains cause lethality. Western Reserve (WR) strain was generated by intracerebral mouse passages, and an intranasal inoculation results in an acute infection of the lung followed by dissemination of the virus to various organs. Intranasal infection with a low dose of WR strain induces an inflammatory infiltrate in the lung, and the virus was cleared 10 to 15 days after infection; however, infection with a high dose of WR strain caused lethality, which has been used as a challenge model to study the effect of antiviral drugs, immune IgG, soluble viral proteins and other vaccine strains. In one report intranasal infection with the WR strain caused pneumonia showing severe alveolar edema and acute necrotizing bronchiolitis and peribronchiolitis as well as neutrophilic infiltrates in the interstitium of the lung. The mechanisms of lethality in mice infected with the lethal dose of WR strain are, however, not well-understood.
In this study, we focused on the differences in virus replication and host immune responses between lethal and non-lethal respiratory infections with VV. We used two VV strains; neurotropic virulent WR strain and the less virulent Wyeth strain. Although BALB/c mice are frequently used for intranasal challenge of vaccinia virus, we used the C57BL6/J strain of mouse in these experiments for two reasons. One is that most knockout mice lacking genes involved in immune responses have been made with C57BL6/J genetic background. The other is that we and one other group have characterized cellular immune responses, especially CD8+ T cell responses, to vaccinia virus in C57BL6/J mice, when this study was planned. Infection of C57BL/6J mouse with a high dose (106 p.f.u. (plaque-forming units)) of the WR strain was lethal, whereas a high dose (106 p.f.u.) of Wyeth strain and a lower dose (104 p.f.u.) of WR strain were not lethal. The WR strain replicated and produced higher titers of virus in the lung and the brain compared to the Wyeth strain. There was, however, no difference between the virus titers in brains of mice infected with the high or low dose of WR strain. Lethal infection with WR strain resulted in fewer lymphocytes and an altered phenotype of T cells in the lung compared to non-lethal infection and uninfected controls, and induced severe thymus atrophy with a marked reduction of CD4 and CD8 double positive (DP) T cells.
Influenza A virus is an RNA orthomyxovirus of approximately 13 kb in length, with an eight-segment genome. It is typically classified on the basis of hemagglutinin (HA) and neuraminidase (NA), of which there are 16 and 9 main variants, respectively (1). Genetic reassortment underpins the potential for transmission between different host species (2) and for the evolution of highly pathogenic variants (3–6), recognized in the WHO list of “ten threats to global health” (7). Seasonal influenza causes an estimated 650,000 deaths globally each year, and the H3N2 variant alone kills 35,000 people each year in the United States (1, 8). Certain groups are particularly at risk, including older adults, infants, young children, pregnant women, those with underlying lung disease, and the immunocompromised (9). The burden of disease disproportionately affects low-/middle-income settings (10). Influenza virus diagnostics and surveillance are fundamental to identify the emergence of novel strains, to improve the prediction of potential epidemics and pandemics (4, 8), and to inform vaccine strategy (11). Diagnostic data facilitate real-time surveillance, can underpin infection control interventions (12, 13), and can inform the prescription of neuraminidase inhibitors (NAI) (9).
Currently, most clinical diagnostic tests for influenza virus depend on detecting viral antigen or on PCR amplification of viral nucleic acid derived from respiratory samples (14). These two approaches offer trade-offs in benefits, as follows: antigen tests (including point-of-care tests [POCT]) are typically rapid but have low sensitivity (15–17), while PCR is more time-consuming but more sensitive (9). Irrespective of the test used, most clinical diagnostic facilities report a nonquantitative (binary) diagnostic result, and the data routinely generated for influenza diagnosis have limited capacity to inform insights into epidemiological linkage, vaccine efficacy, or antiviral susceptibility. On these grounds, there is an aspiration to generate new diagnostic tests that combine speed (incorporating the potential for POCT [18, 19]), sensitivity, detection of coinfection (20, 21), and generation of quantitative or semiquantitative data that can be used to identify drug resistance and reconstruct phylogeny to inform surveillance, public health strategy, and vaccine design.
The application of Oxford Nanopore Technologies (ONT) sequencing to generate full-length influenza virus sequences from clinical respiratory samples can address these challenges. ONT offers a “third-generation,” portable, real-time approach to generating long-read single-molecule sequence data, with demonstrated success across a range of viruses (20, 22–24). To date, Nanopore sequencing of influenza virus has been reported using high-titer virus from an in vitro culture system, producing full-length genome sequences through direct RNA sequencing (25), or using targeted enrichment by either hybridization of cDNA (26) or influenza virus-specific PCR amplification (27).
We therefore aimed to optimize a metagenomic protocol for detecting influenza viruses directly from clinical samples using Nanopore sequencing. We determine its sensitivity compared to that of existing diagnostic methods and its accuracy compared to short-read (Illumina) sequencing, using clinical samples from hospital patients during an influenza season and samples from a controlled laboratory infection in ferrets. Further optimization is required before the Nanopore method can be rolled out as a diagnostic test, but we highlight the potential impact of this technology in advancing molecular diagnostics for respiratory pathogens.
Humans can become infected with CCHFV via tick bites and butchering of infected livestock and in the health-care setting during the care of infected patients
Figure 1). Following an incubation period of a few days, the initial symptoms of CCHF are a non-specific febrile illness that can occur suddenly. Sudden onset of fever, myalgia, diarrhea, nausea, and vomiting is typically reported. After this, patients enter the hemorrhagic period in which they begin exhibiting hemorrhages at various sites around the body
8. Case fatality rates can differ between outbreaks but typically range from 5% to 30%
3. However, subclinical or mild cases of CCHF may go unnoticed and may represent a substantial portion of CCHFV infections in humans
9. Despite the known genetic diversity of CCHFV, whether the infecting strain of CCHFV influences disease severity and outcome is unknown. High viral loads, absence of early antibody responses, and high levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are common predictors of poor outcome
Figure 1). Thrombocytopenia and prolonged clotting times are also seen in severe cases
15. Levels of inflammatory cytokines are elevated in severe and fatal CCHF cases
19, suggesting that CCHFV infection induces an inflammatory immune response.
The diagnosis of suspected CCHF cases can be accomplished by using reverse transcription–quantitative polymerase chain reaction (RT–qPCR) during the viremic phase of disease. RT–qPCR can also determine viral load, which often is correlated with disease outcome. An important consideration for these assays is the substantial genetic diversity of CCHFV; however, assays that can recognize a multitude of CCHFV genotypes have been developed
22. Enzyme-linked immunosorbent assay and indirect immunofluorescent assay for the detection of human IgM and IgG CCHFV-specific antibodies are approaches of choice for serological diagnosis, and commercial kits are available. These tests may not be appropriate for suspected cases early during the acute phase of disease, as antibody responses are often absent or delayed in serious CCHF cases.
Amongst pathogens, RNA viruses were a major source of emerging diseases during the last 30 years. High mutation rate and in case of segmented genome, reassortment are responsible for genetic adaptability and variability of these viruses.
Two pathogens affecting cattle and sheep were responsible for major outbreaks in Mainland Europe in the past 15 years: Bluetongue virus (BTV) and Schmallenberg virus (SBV). These outbreaks were singular in several ways: the diseases were previously either never reported in such northern locations (bluetongue virus) or recently discovered (Schmallenberg virus); their emergence still has unexplained aspects; both viruses displayed the ability to cross the placental barrier. Moreover, these events confirmed that palearctic endemic Culicoides species contribute to the spread of BTV and SBV and to the epizootic aspect of the diseases.
Bluetongue virus causes the eponymous bluetongue disease (BT). BTV belongs to the family Reoviridae, subfamily Sedoreovirinae, and represents the type specie of the Orbivirus genus. The family Reoviridae currently contains fifteen genera of multi-segmented dsRNA viruses, including pathogens of a wide range of vertebrates (including humans), arthropods, plants, and fungi. Unlike the other reoviruses, all orbiviruses are arthropod-borne viruses (arboviruses). This genus currently contains 22 species as well as 10 unclassified “orbiviruses”.
Until recent nomenclature changes implemented by the International Committee on Taxonomy of Viruses Schmallenberg virus was part of the Bunyaviridae family, genus Orthobunyavirus, grouped within the serogroup Simbu along with at least 27 other virus species. The members of the Simbu serogroup show cross-reactions to the complement fixation test but are distinguished by seroneutralization and by genetic sequence analysis. Yet still part of the Orthobunyavirus genus, SBV, AKAV and Aino virus (AINOV) are now considered exemplar viruses of the species Sathuperi orthobunyavirus, Akabane orthobunyavirus, and Shuni orthobunyavirus, respectively. These belong to the new order Bunyavirales, family Peribunyaviridae (formerly Bunyaviridae), which comprises the genus Orthobunyavirus and Herbevirus (host range limited to insects).
Despite their belonging to different viral families, BTV and SBV have several features in common. These converging aspects warrant the present work discussing more specifically the elements to consider while designing experimental infections targeting ruminant host species. A particular emphasis will be given to placental crossing and teratogenic potential of these two viruses.
Chlamydiae are implicated in a wide variety of diseases in both animals and humans. Although acute infections in animal chlamydioses are the most commonly reported, chronic chlamydial infections are also associated with a variety of diseases in humans and animals. These latter infections are characterized by inflammation and scarring resulting in significant damage of the host. A causative role in chronic diseases requires that chlamydiae persist within infected tissue for extended periods of time. Current theories, based primarily on in vitro data, suggest that chlamydial persistence, and the resulting chronic inflammation, is linked to morphological and metabolic conversion of the actively replicating and intracellular reticulate body (RB) into an alternative, non-replicative form known as an aberrant body (AB). In vitro, alterations of the normal developmental cycle of Chlamydia trachomatis and Chlamydia pneumoniae can be induced by Interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and penicillin G exposure as well as amino acid or iron deprivation and monocyte infection. To date, in vitro models for animal pathogens, Chlamydia abortus and Chlamydia pecorum have not been described although both organisms are associated with chronic disease in koalas and small ruminants.
In pigs, several chlamydial species, including Chlamydia abortus, Chlamydia psittaci, Chlamydia pecorum and Chlamydia suis, have been implicated in a variety of disease conditions including conjunctivitis, pneumonia, pericarditis, polyserositis, arthritis, abortion and infertility. In the gastrointestinal tract, chlamydiae appear to be highly prevalent but only occasionally cause enteritis. They have been found in the intestine of diarrheic and healthy pigs and could be demonstrated in mixed enteric infections. Pospischil and Wood first described an association between Chlamydiaceae and lesions in the intestinal tract of pigs and assumed a synergistic effect in co-existence with Salmonella typhimurium. Further, mixed infections with Eimeria scabra, cryptosporidia, and porcine epidemic diarrhea virus (PEDV) have been described in the past. PEDV, a member of the family Coronaviridae, is a well-known cause of diarrhea in pigs. After the identification of PEDV in 1978 by Pensaert and Debouck, more than a decade passed before the virus could be adapted for propagation in cell cultures. Examination of infected Vero cell cultures by direct immunofluorescence revealed single cells with granular cytoplasmic fluorescence as well as formation of syncytia with up to 50-100 nuclei or more. Typical features of syncytial cells were growth, fusion and detachment from cell layers after they had reached a certain size. Biomolecular studies revealed major genomic differences between cell culture-adapted (ca)-PEDV and wild type virus.
Cell culture model of co-infection with ca-PEDV and Chlamydia has been established recently to investigate the interaction of ca-PEDV and Chlamydiaceae in mixed infections and to detect possible synergistic or additive effects of possible significance in clinical enteric disease in pigs. In that study, abnormally large chlamydial forms were observed in dually infected cell layers by immunofluorescence suggesting that ca-PEDV co-infection might alter the chlamydial developmental cycle in a manner similar to that observed during persistent infections. To confirm these initial observations, we established a cell culture model of mixed infections with Chlamydia and a cell culture-adapted porcine epidemic diarrhea virus (ca-PEDV) and hypothesized that this would result in the generation of persistent chlamydial forms. This data demonstrates that ca-PEDV co-infection, indeed, alters the developmental cycle of Chlamydia pecorum and Chlamydia abortus in a similar manner to other inducers of chlamydial persistence.
Adult female C57BL6/J mice were inoculated with various doses of the WR and Wyeth strains intranasally. Weight change and survival of infected mice were recorded daily. Inoculation with higher doses (106 p.f.u. and 105 p.f.u.) of the WR strain induced rapid and severe weight loss, which became obvious at 3 days post-infection (Fig. 1A), and most of mice died at 7–10 days post-infection (with 106 p.f.u. all mice died by 8 days post-infection) (Fig. 1B). Lower doses (104 p.f.u. and 103 p.f.u.) of the WR strain caused mild weight loss, and all mice survived. These mice recovered their weight after 6–8 days post-infection (Fig. 1A). The 50% lethal dose (LD50) of WR strain was calculated as 4.2 × 104 p.f.u., which is similar to the LD50 reported for BALB/c mouse. Wyeth strain did not kill mice (Fig. 1B) or cause weight loss (Fig. 1A) even when 106 p.f.u. of the virus was inoculated.
Streptococcus equi subspecies equi is the causative agent of strangles, a highly contagious upper respiratory tract infection of horses. Typical clinical signs of disease include fever, inappetance, lethargy, submandibular or retropharyngeal lymphadenopathy or purulent drainage, or purulent nasal discharge. Complications of S. equi infection can occur and include airway obstruction from lymphadenopathy, disseminated abscesses from hematogenous spread, or purpura hemorrhagica and various diseases caused by immune‐mediated processes.1, 2, 3, 4, 5
Streptococcus equi M protein (SeM) antibody titers are typically measured to determine if a horse has developed a complication of strangles, such as purpura hemorrhagica or metastatic abscess formation, or to determine if a horse is at risk of purpura hemorrhagica if they were to be vaccinated. Both the 2005 and 2018 American College of Veterinary Internal Medicine consensus statements on strangles state that a very high titer (≥1:12 800) is associated with metastatic abscess formation or purpura hemorrhagica and that high titers (1:3200‐1:6400) are detected 4‐12 weeks after infection.1, 2 Anecdotally, horses can have high titers (≥1:12 800) 4‐8 weeks after infection and no signs of complications (authors' personal observations, KMD, LAB, ACT). The objective of this study was to measure SeM antibody titers on horses after outbreak to determine if titers detect the presence of complications.2 An additional objective was to follow SeM antibody titers out to 7 months after infection to determine immunoglobulin decay and to monitor for development of additional complications. We hypothesized that the magnitude of SeM antibody titer after infection (SeM titer ≥1:12 800) will be useful to monitor for the presence of complications or for the risk of development of complications.
It is estimated by the World Health Organization (WHO) that approximately 36.9 (34.3~41.9) million persons worldwide are infected with human immunodeficiency virus (HIV), which is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS), including 1.7 million in the United States. Influenza A virus can cause acute respiratory infection in humans and animals throughout the world, and has continued to be a significant public health threat, leading to substantial global morbidity and mortality and an average of approximately 23,600 deaths annually in the United States alone.
The impact of influenza virus on HIV infection has not been well investigated. Little is known about influenza virus infection in HIV-positive individual. HIV infection has been shown to be related to worse prognosis of influenza. HIV-infected patients in Canada who also had pandemic 2009 influenza A (H1N1) virus (pH1N1) infection had more severe illness than those who did not have the co-infection, and fatality was higher than for patients who were not co-infected in California (USA). Recent reports indicate that adults with AIDS experience substantially elevated influenza-associated mortality.
Influenza virus infection has been associated with viremia in human and animal models. Viral RNA has been detected in blood in severe human pH1N1 infection. Encapsidated pH1N1 RNA is stable in blood derived matrices and influenza viruses can be transmitted by blood transfusion in ferrets. Normally, influenza A virus infection is confined to the airways where the virus replicates in respiratory epithelial cells. Cumulated reports indicate that influenza viruses can infect and replicate in blood cells, such as dendritic cells, primary monocytes/macrophages, and T cells.
Infection with HIV-1 can result in apoptotic cell death through activation of both death receptor-mediated and Bax/mitochondrial-mediated apoptotic pathways, which cause a progressive depletion of a select group of immune cells namely the CD4+ T helper cells leading to immunodeficiency. While HIV directly and selectively infects CD4+ T cells, the low levels of infected cells in patients is discordant with the rate of CD4+ T cell decline and argues against the role of direct infection in CD4 loss. A viral protein, neuraminidase (NA), derived from the human influenza virus was reported to enhance the level of HIV-1-mediated syncytium formation and HIV-1 replication. However, it is not known whether influenza A virus in blood affects HIV-1 replication, or reactivates HIV-1 replication in HIV-1-infected cells. Here, we showed that pandemic influenza A (H1N1) virus infection increased apoptotic cell death and HIV-1 replication in HIV-1 infected Jurkat cells.
Viral hemorrhagic fevers (VHF) constitute a group of clinically similar diseases characterized by mild to severe febrile acute syndromes with vascular damage, plasma leakage and bleeding. The most clinically prevalent human hemorrhagic fever viruses belong to the families Arenaviridae, Filoviridae, Hantaviridae, and Flaviviridae, which share some structural and replicative characteristics (Figure 1), as well as some immunity issues in the host. Indeed, the families Arenaviridae and Hantaviridae now belong to the same order: Bunyavirales. However, the pathogenesis and role of the immune system in the development of mild or severe disease, and the protective or pathogenic function of some components of the immune system are still not clear. Particularly, the contribution of T-cell mediated immunity in disease protection and pathogenesis is yet to be characterized, especially the important associations between the magnitude of the T-cell response with disease survival or disease exacerbation. Thus, apparently, T-cells play a context-dependent role during VHF that is necessary to define in order to improve the patient clinical outcome and to develop vaccines or immunotherapeutic strategies. Here, we discuss the role of T-cells in the control of VHF and disease pathogenesis and their potential contribution to vaccine development. T-cell response features of representative viruses from each family, such as lassa virus (LASV) for arenavirus, ebola virus (EBOV) for filovirus, Hantaan Virus (HTNV) for hantavirus, and dengue virus (DENV) for flavivirus, are addressed. Since studies on vaccination against yellow fever virus (YFV) have largely contributed to the understanding of the dynamics of human T-cell differentiation after viral infections, and a large body of evidence supports the role of T-cells in the effectiveness of this vaccine, here we also discuss the T-cell response after YFV vaccination.
Medical procedures that have the potential to create aerosols in addition to those that patients regularly form from breathing, coughing, sneezing, or talking are called AGMPs. While there are many suspected AGMPs, few AGMPs were confirmed to generate aerosols. In order to determine which AGMPs could be important for nosocomial virus transmission, we first need to characterize what aerosols are and how they are created.
Aerosols are particles suspended in air that can contain a variety of pathogens, including viruses, and there is ongoing debate about how to classify them. Many divide aerosols into the categories of small droplets (which some exclusively call aerosols) and large droplets, with small droplets having the potential to desiccate and form droplet nuclei that travel long distances, while large droplets do not evaporate before settling on surfaces. Classifying aerosols by their initial size is relevant in relation to their dispersal patterns, but it is also important to classify aerosols according to where they deposit in the respiratory tract because pathogenesis can be influenced by whether a virus deposits in the upper respiratory tract (URT) or lower respiratory tract (LRT). Dispersal and deposition depend on a variety of factors, and there is no exact cutoff for small and large droplets. Some authors use ≤5 µm in diameter as a cutoff for small droplets, while another possible cutoff between aerosol types is 20 µm, since aerosols ≤20 µm in diameter can desiccate to form droplet nuclei, and aerosols ≥20 µm do not deposit substantially in the LRT.
Often the term airborne transmission is used to describe infection by small droplet aerosols and droplet nuclei, while droplet transmission refers to the route of large droplet aerosols. Since aerosols can be of multiple sizes, we use the term aerosol transmission to generally describe transmission through the generation of infectious small and large droplet aerosols. In addition to these modes of transmission, AGMPs may also create opportunities for direct contact and fomite transmission, which may be difficult to distinguish.
HCWs are considered to be at risk for nosocomial virus transmission from both small and large droplet aerosols, for both seem to play a role in human-to-human virus transmission. Small droplets can be inhaled into the LRT, while large droplets can splash into the eyes or mouth and deposit in the URT. Certain respiratory viruses, like influenza A virus, are believed to transmit between people by both small and large droplets, whereas other nonrespiratory viruses, like EBOV, could theoretically be spread by large droplets because small droplets containing these viruses are not known to form in the human respiratory tract. It is unknown whether certain AGMPs generate either small or large droplets, or both. Therefore, depending on what aerosols are formed, AGMPs could potentially amplify a normal route of transmission for respiratory viruses or open up a new route of transmission for other viruses.
We can group possible AGMPs into two categories: procedures that mechanically create and disperse aerosols and procedures that induce the patient to produce aerosols (Figure 1 and Table 1). Procedures that irritate the airway, such as bronchoscopy or tracheal intubation, can cause a patient to cough forcefully, potentially emitting virus-laden aerosols, and both of these procedures are associated with the possibility of increasing the risk of SARS-CoV transmission among HCWs. The pressure on a patient’s chest during cardiopulmonary resuscitation can also induce a “cough-like force”, which was another possible source of SARS-CoV nosocomial transmission. Sputum is also routinely collected from patients for diagnostic purposes by cough induction, but it is not associated with nosocomial virus transmission.
In contrast to causing a patient to produce aerosols, AGMPs can also mechanically create and disperse respiratory aerosols through procedures such as ventilation, suctioning of the airway, or nebulizer treatment. Both manual ventilation, using a bag-valve-mask, and other forms of noninvasive ventilation (NIV), such as continuous positive airway pressure (CPAP), bilevel positive airway pressure (BiPAP), and high-frequency oscillatory ventilation (HFOV) are associated with SARS-CoV nosocomial transmission. Although the exact mechanisms of how these procedures create virus-laden aerosols in the respiratory tract remain unknown, it is possible that forcing or removing air from the respiratory tract could generate aerosols.
While AGMPs are traditionally thought of in regard to the generation of respiratory aerosols, AGMPs can also aerosolize infected fluids in other regions of the human body. Surgical techniques can aerosolize blood and possibly viruses. For example, infectious HIV-1 was found in the aerosols generated by surgical power tools, and a tracheotomy was associated with SARS-CoV transmission. Lasers can create plumes of debris that contain infectious aerosolized virus, as well. It is important to recognize the range of AGMPs and the circumstances under which they might be performed on infected patients. In order to associate certain AGMPs with nosocomial virus transmission, researchers need to test whether certain procedures generate aerosols with infectious virus, either through hospital sampling or laboratory procedures.
Given its continuous exposure to the outside environment, the respiratory mucosa is highly susceptible to viral infection. The human respiratory tract can be infected with a variety of pulmonary viruses, including respiratory syncytial virus (RSV), influenza virus, human metapneumovirus (HMPV), rhinovirus (RV), coronavirus (CoV), and parainfluenza virus (PIV) (1). The severity of disease associated with respiratory viral infection varies widely depending on the virus strain as well as the age and immune status of the infected individual. Symptoms can range from mild sinusitis or cold-like symptoms to more severe symptoms including bronchitis, pneumonia, and even death. RSV is the leading cause of severe lower respiratory tract infection in children under 5 years of age (2). RSV is commonly associated with severe lower respiratory tract symptoms including bronchiolitis, pneumonia, and bronchitis and is a significant cause of hospitalization and mortality in children, the elderly, and immunocompromised individuals (2–6). Similarly, PIV commonly infects children and is a major cause of croup, pneumonia, and bronchiolitis (7, 8). Seasonal influenza infections, most often of the influenza A virus (IAV) subtype, are responsible for 3–5 million cases of severe infection annually (9). Seasonal IAV infections also result in approximately 290,000–650,000 deaths per year, most commonly in either young children or elderly populations (9–11). However, infection with emerging pandemic IAV strains, such as the 2009 H1N1 pandemic strain, primarily induces severe disease and mortality in otherwise healthy adults younger than 65 years of age (12). In contrast, respiratory infection with HMPV, RV, and CoV are most commonly associated with symptoms of the common cold (13–15). Two notable exceptions are severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome (MERS) CoV, which cause acute respiratory distress and mortality in infected individuals (16–18).
Despite their profound impact on human health, most common respiratory viruses lack an approved vaccine. The strategy employed most often in vaccine development is the induction of robust neutralizing antibody responses. However, the hallmark of many respiratory viral infections, including RSV, HMPV, and RV, is the ability for reinfections to occur frequently throughout life (19–21). This suggests that the antibody response to these respiratory viruses may wane over time. Indeed, despite a correlation between pre-existing nasal IgA and protection from reinfection, the development of long-lasting RSV- and RV-specific mucosal IgA responses was poor in infected adults (22, 23). Although IAV-specific neutralizing antibodies are elicited efficiently through either infection or vaccination, IAV vaccine formulations must be redeveloped annually to account for the rapid mutations of HA and NA genes in seasonal strains (24). Therefore, vaccinations that solely promote the induction of neutralizing antibodies may not be optimal in providing protection against many respiratory virus infections. The induction of cellular immune responses has thus far received little attention in respiratory virus vaccine development. CD8 T cells play a critical role in mediating viral clearance following many respiratory virus infections including RSV, IAV, and HMPV (25–27). In addition, recent murine studies utilizing CD8 T cell epitope-specific immunization strategies observed significantly reduced lung viral titers following IAV, RSV, or SARS challenges (28–30). Therefore, the induction of virus-specific CD8 T cell responses has the potential to improve upon the efficacy of current vaccination strategies. Here, we review the current literature on CD8 T cell responses following respiratory virus infections and discuss how this knowledge may best be utilized in the development of future vaccines.
In recent years, the repeated outbreak of hantavirus disease has caused a serious threat to human health. The spread of hantavirus from natural hosts to humans is a natural ecological process; however, the outbreak of hantavirus is driven by striped field mouse population cycle dynamics and seasonal climate change (Tian and Stenseth, 2019).
Hantavirus is a virus transmitted mainly by rodent animals, mainly through urine, feces, and saliva and the aerosols produced by them, but rarely by the bites of infected animals (Brocato and Hooper, 2019). In recent years, the infection rate of hantavirus has increased in China and Europe (Dong et al., 2019). Hantavirus disease has turned out to be a newly identified but not a “new” disease in Germany (Kruger et al., 2013). The clinical presentations may vary according to viral strains prevalence in different regions. In Asia, hantavirus infection by Hantan virus (HTNV) and Seoul virus (SEOV) targets mainly the human kidney and causes hemorrhagic fever with renal syndrome (HFRS). In North America, infection by Andes virus (ANDV) and Sin Nombre virus (SNV) manifests in mainly the lung and leads to hantavirus pulmonary syndrome (HPS) or hantavirus cardiopulmonary syndrome (HCPS), with high mortality rates; in Europe, infection by Puumala virus (PUUV) and Dobrava-Belgrade virus (DOBV) typically causes a milder form of HFRS, nephropathia epidemica (NE) (Echterdiek et al., 2019).
Currently, there is no approved post-exposure therapeutic countermeasure against hantaviral infection, but diversified treatment strategies have been developed and applied to manage HFRS or HCPS. These strategies target viral life cycle, host immunological factors, or patient clinical symptoms. Preventive measures against hantaviral infection, especially vaccine development, are essential for future pandemics. In this paper, we reviewed the epidemiology and pathogenesis of hantavirus, and discuss the existing knowledge on vaccine and therapeutics against these diseases in order to shed light on the development of new vaccines and treatments.
Crimean–Congo hemorrhagic fever virus (CCHFV) is a negative-sense RNA virus in the
Nairoviridae family within the
Bunyavirales order of viruses. CCHFV contains three genomic segments: small and medium, which encode for the nucleoprotein and glycoproteins, respectively, and a large segment encoding the RNA-dependent RNA-polymerase. CCHF as a disease was first described in humans in the 1940s when soldiers re-occupying abandoned farmland in the Crimea became ill with a hemorrhagic disease
1. In the late 1960s, it was discovered that the causative agent of this hemorrhagic disease in the Crimea was similar to the causative agent of hemorrhagic disease in the Belgian Congo (current Democratic Republic of the Congo)
2, and the name “Crimean–Congo hemorrhagic fever virus” was ascribed to the pathogen. The main vector and reservoir of CCHFV are hard-body ticks principally of the
Hyalomma genus, although there is limited evidence that other species of ticks such as
Dermacentor species may be vectors
3. Vertebrate hosts such as domestic livestock and wild animals such as hares likely serve as amplifying hosts of CCHFV, with uninfected ticks becoming infected during feeding on viremic animals or during co-feeding with infected ticks
Figure 1). The
Hyalomma vector is found throughout Africa, Southern and Eastern Europe, the Middle East, India, and Asia and cases of CCHF are reported throughout these regions
7; an estimated 10,000 to 15,000 human infections with CCHFV occur each year, although most of these are subclinical and unrecognized
7. In correlation with the extensive geographic distribution of CCHFV, CCHFV exhibits substantial genetic diversity among geographically distinct isolates; isolates differ at the amino acid level by 5% in the nucleoprotein and L protein and up to 25% in the glycoprotein precursor
Vaccinia virus (VV), a member of the Poxviridae family, is an enveloped, DNA virus with a genome of 192 kb encoding about 200 proteins. Various cell lines can be infected by VV, including HeLa, CV-1, mouse L, and chicken CEF cells. VV causes major changes in host cell machinery shortly after infection, and cytopathic effects (CPE) are observed several hours after infection with VV. VV infection modulates host cell gene expression: several previous studies have shown that mRNA synthesis in the host cells was inhibited immediately after VV infection. Microarray analysis showed that around 90% of the host genes were down-regulated after VV infection, including genes involved in DNA replication, transcription, translation, apoptosis, and the proteasome-ubiquitin degradation pathway. Only a smaller fraction of host genes were up-regulated after VV infection, including WASP protein, and genes implicated in immune responses.
Several viral factors of VV utilize ATP and several steps in viral multiplication of VV require ATP. ATP is also required for DNA packaging and capsid maturation of herpes simplex virus, for capsid assembly and release of type D retrovirus, for capsid assembly of human immunodeficiency virus, and for budding of influenza virus. Therefore, it was expected that viral factors would modulate cellular energetics to benefit the virus, though this area is understudied.
In this study, the possible up-regulation of host cell genes after VV infection was analyzed by differential display-reverse transcriptase-polymerase chain reaction (ddRT-PCR), a simple technique with high sensitivity and specificity . Two mitochondrial genes involved in the electron transport chain (ND4 and COII) to generate ATP were found to be up-regulated after VV infection using this assay.
Viruses are a global source of significant health and economic burdens. Interactions between humans and animals are the paramount driving force for the ever-growing distribution and emergence of novel viruses. Other reasons for the spread of new viruses include climate change, globalization, social mobilization and intensive farming1–3. A novel virus might emerge in the form of an outbreak, and in this scenario, rapid diagnosis is of extreme importance for the selection of prevention and treatment strategies. However, such a diagnosis is not trivial, especially for RNA viruses, since the viruses in this category have small but highly variable genomes4.
Viruses can be identified by a wide range of techniques. Traditional methods rely on morphological characteristics observed by light microscopy or transmission electron microscopy (TEM) in various specimens such as cell cultures and fertilized eggs. Serology, as well as antibody-based diagnostics, allow to identify the virus and in some cases even at the species level. However, these methods provide only morphological clues, depend on the availability of an antiserum and, for the most part, are not strain specific5. In recent decades, molecular methods such as PCR, RT-PCR and microarrays, which are in most cases more sensitive than traditional techniques, have been used to complement and even replace traditional techniques. However, most of the molecular assays mentioned above are designed to be pathogen specific or are aimed at a limited group of infectious agents. The narrow scope of these methods significantly limits their ability to discover new, unknown pathogens and hampers our ability to reveal the full diversity of a given clinical specimen6,7.
High-throughput sequencing (HTS) technologies, developed in recent years, have substantially improved the capability of comprehensive detection of pathogens without any prior assumptions about the characteristics of the organisms (i.e., “unknown” samples). These massive parallel sequencing platforms can sequence mixtures of genetic materials from heterogeneous samples with high sensitivity and speed and at a lower cost per base than traditional Sanger sequencing8,9. In addition, HTS technologies, have benefits other than the improved detection of known and unknown pathogens in different samples. Among these benefits is the ability to detect nonculturable organisms as well as coinfection, drug resistance or response to therapy10,11.
The use of HTS for characterization of unknown viral pathogens in relevant clinical samples remains limited, primarily because of the large ratio between the genome sizes of hosts and pathogens. This limitation is especially true of blood-related samples, where white blood cells are abundant. Nonetheless, virus detection in clinical samples by HTS is starting to be increasingly used. Studies that have tried to identify unknown RNA viruses by using HTS have been conducted mostly in less-relevant clinical samples (i.e., brain) and provided diagnoses in shorter time frames because they did not require culture first, while other studies have typically obtained between tens to a few thousands of viral reads, a number that might be sufficient for resequencing against a model organism but not for characterizing novel viruses12–20.
In this study, an HTS-based approach was applied to identify the origin of a viral pathogen found to be present in plasma obtained from a hyperimmune horse21. To this end, we have established a straightforward procedure that includes virus enrichment in cell culture followed by RNA extraction from the growth medium, rapid library preparation, sequencing and in-depth data analyses. By following this procedure, we successfully identified and characterized a novel species belonging to the Orthobunyavirus genus.
EV-D68 preferentially causes severe respiratory symptoms in children and adults that have a prior history of asthma. Thus, in addition to naïve mice, HDM-sensitized and -challenged mice also been studied. In mice with allergic airways disease, EV-D68 enhances allergen-induced type 2 inflammation with increased expression of lung IL-5, IL-13 and Muc5ac and augmentation of bronchoalveolar lavage fluid eosinophils and airway responsiveness.
We collected respiratory samples from the clinical microbiology laboratory at Oxford University Hospitals NHS Foundation Trust, a large tertiary referral teaching hospital in Southeast England. We worked with anonymized residual material from throat and nose swabs generated as a result of routine clinical investigations between January and May 2018. Samples were collected using a sterile polyester swab inoculated into 1 to 3 ml of sterile viral transport medium (VTM), using a standard approach described on the CDC website (28). During the study, respiratory samples submitted to the clinical diagnostic laboratory were routinely tested by a PCR-based test using the GeneXpert assay (Cepheid) to detect influenza A and B viruses and respiratory syncytial virus (RSV). The workflow is shown in Fig. 1. Samples from patients in designated high-risk locations (hematology, oncology, and critical care) were tested using the BioFire FilmArray (bioMérieux) to detect an expanded panel of bacterial and viral pathogens. Quantitative data (cycle threshold [CT]) were generated by the GeneXpert assay, and we used the influenza virus CT value to estimate the viral titers in clinical samples. Using the GeneXpert assay, up to 40 PCR cycles are performed before a sample is called negative (i.e., positives have a CT value of <40). Quantification was not available for the BioFire results.
For methodological assessment, we focused on four categories of samples, as follows: positive pool, negative pools, individual positive samples, and individual negative samples. For the positive pool, we pooled 19 throat swab samples that had tested positive for influenza A virus in the clinical diagnostic laboratory to provide a large enough sample to assess reproducibility (Fig. 1B). For the negative pools, we generated three pools of throat swab samples that had tested negative for influenza virus (consisting of 24, 38, and 38 individual samples) (Fig. 1B). For the individual positive samples, we included 40 individual samples (35 throat swabs and 5 nasal swabs) that had tested positive for influenza A or B virus, selected to represent the widest range of GeneXpert assay CT values (13.5 to 39.3; valid test result range, 12 to 40). For the individual negative samples, we selected 10 individual throat swab samples that were influenza virus negative.
Feline immunodeficiency virus (FIV) is a lentivirus closely related to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) that naturally infects numerous wild and domestic feline species. Individual feline species typically harbor genetically-distinct species-specific FIV strains [1–3]. Infection with the domestic cat (Felis catus) strain of FIV (FIVFca) results in CD4+ T-cell depletion and pathogenic disease which progresses to AIDS-like immune dysfunction and ultimately death. FIV infection and disease in domestic cats bears many similarities to HIV infection and AIDS in humans including similar routes of infection, cell and tissue tropism, clinical symptoms and course of disease. Thus, FIV infection of the domestic cat is a useful animal model for studying HIV infection and vaccine development. In contrast, FIV infection of nondomestic felid species has little measurable impact on survival of the natural host [2,10–13], but may be associated with long-term immune cell depletion and other disease sequelae [14–17]. Cross-species transmission events are thought to be limited by lack of contact between host species and the action of host-specific cellular restriction factors. Experimental infection of domestic cats with a lentivirus (puma lentivirus (PLV) or FIVPco) native to the cougar (Puma concolor) results in productive yet avirulent infection with no detectable T-cell depletion or clinical disease, providing a model to evaluate mechanisms for restriction of lentiviral cross-species infection. PLV viral load diminishes over time to virtually undetectable levels in circulation and lymphoid tissues with low level infection of the gastrointestinal tract. Intensive sequence analysis of PLV genomes integrated in cat blood cells showed a strong bias toward G-to-A hypermutation, a hallmark of cellular cytidine deaminase-mediated viral restriction [22–25], suggesting that cellular restriction likely plays a role in control of PLV infection in domestic cats. Thus PLV infection is a model for cross-species transmission of a virus which is not well-adapted to long-term replication in the new host and remains nonpathogenic.
Previously we have studied whether infection of domestic cats with PLV can impart resistance to subsequent infection with virulent FIV. Cats infected with PLV 28 days prior to FIV challenge were protected from the marked CD4+ T-cell depletion experienced by cats challenged with FIV alone. PLV/FIV co-infected cats had a unique immunologic profile distinct from FIV single infected cats—including elevated levels of CD8, CD25 and FAS expressing cells and elevated expression of the cytokines IL-4 and IFNγ. In contrast, we did not detect evidence of adaptive immunity to FIV such as neutralizing antibodies or virus-specific cytotoxic T-cells. These data suggest that innate rather than adaptive immune mechanisms are associated with PLV-induced protection from FIV disease. Additionally, examination of FIV population genetics demonstrated that during PLV/FIV co-infection FIV underwent a population bottleneck at approximately three weeks post-infection not observed in FIV single infection. The nature of this bottleneck remains to be determined, but the data are consistent with PLV-induced host restriction of normally virulent FIV. Collectively, these studies suggest that host innate immunity has a role in mediating PLV-induced modulation of FIV infection and disease. However, the exact nature of PLV-induced protection remains to be elucidated.
Our previous studies aimed at determining the mechanism(s) of PLV-induced protection from FIV disease have focused largely on timepoints early after FIV infection (<120 days post-PLV infection) using peripheral blood mononuclear cells (PBMC) for analysis. In this study we focused on PLV and FIV infection and innate immune response in multiple infected cat tissues at a later chronic infection timepoint (159 days post-PLV infection) for the purpose of detecting changes in infection and immunity which may be specific to important tissue reservoirs of infection and provide insight into PLV mediated protection. Further, evaluating distribution of both provirus and viral RNA transcription across a suite of tissues allows analysis of the hypothesis that PLV infection alters the distribution and tissue reservoirs of susbsequent FIV infection.
We found that PLV co-infection altered the magnitude and variability of FIV infection and innate immunity in tissues compared to FIV single infection—to a remarkable extent considering our analyses were conducted nearly five months following co-infection. While somewhat surprisingly, FIV proviral load tended to be higher in tissues from co-infected versus single-infected animals, viral transcripts tended to be lower in the co-infection state. Comparison of FIV proviral load and FIV mRNA load among single-infected versus co-infected tissues indicated that PLV co-infection limits FIV productive infection (viral mRNA expression) relative to FIV proviral load. Collectively, these data suggest that PLV-induced protection from FIV disease may be at least partially mediated by persistent alterations of innate immunity resulting in limitation of FIV productive infection. It is also possible that restriction of target cell populations via PLV-induced immune activation or alteration of susceptibility for other reasons results in an altered FIV infection landscape. This hypothesis is supported by the finding that viral and cytokine transcription rates were more variable during single FIV infection, and as reported previously, FIV replication in the face of previous PLV infection is highly constrained during acute infection. If during co-infection, FIV is restricted to a cell type with longer half-life that is less permissive for viral replication, we would predict an outcome similar to that observed in this study. These experiments pose a new paradigm for assessment of protective immunity against HIV/AIDS—namely that perturbation of early innate immune parameters and circulating cell phenotype can alter the outcome of a virulent lentiviral infection.
Since the beginning of modern virology in the 1950s, transmission electron microscopy (TEM) has been one of the most important and widely used techniques for the identification and characterization of new viruses. Two TEM techniques are usually used for this purpose: negative staining on an electron microscopic grid coated with a support film and (ultra) thin section TEM of infected cells, fixed, pelleted, dehydrated, and embedded in epoxy plastic. Negative staining can be conducted on highly concentrated suspensions of purified virus or cell culture supernatants. For some viruses, TEM can be conducted on contents of skin lesions (e.g., poxviruses and herpesviruses) or concentrated stool material (rotaviruses and noroviruses). For successful detection of viruses in ultrathin sections of infected cells, at least 70% of cells must be infected, and so either high multiplicity of infection (MOI) or rapid virus multiplication is required.
Viruses can be differentiated by their specific morphology (ultrastructure): shape, size, intracellular location or, for some viruses, from the ultrastructural cytopathology and specific structures forming in the host cell during virus replication. Usually, ultrastructural characteristics are sufficient for the identification of a virus at the level of a family. In certain cases, confirmation can be obtained by immuno-EM performed either on virus suspension before negative staining or on ultrathin sections. This requires virus-specific primary antibodies, which might be not available in the case of a novel virus. For on-section immuno-EM, OsO4 post-fixation must be omitted and the partially dehydrated sample must be embedded in a water-miscible acrylic plastic (usually LR White). The ultrastructure of most common viruses is well documented in good atlases and book chapters and many classical publications of the 1960s, 1970s, and 1980s. Several excellent reviews were recently published on the use of TEM in the detection and identification of viruses.
West Nile virus (WNV), a plus-sense, single-stranded neurotropic flavivirus, has been a public health concern in North America for more than a decade. The virus is maintained in an enzootic cycle that involves mosquitoes and birds, with humans and horses as incidental hosts. Infection in humans results from mosquito bites, blood transfusion, organ transplantation, breast feeding, and in utero or occupational exposure. WNV infection of the central nervous system (CNS, neuroinvasive disease) commonly presents as encephalitis, meningitis, or acute flaccid paralysis. The overall mortality rate in persons who develop WNV neuroinvasive disease is about 10%, although the mortality rate increases significantly in the elderly and immunocompromised. Recently, some WNV convalescent patients were reported to have significant long-term morbidity years after their acute illness; symptoms include muscle weakness and pain, fatigue, memory loss, and ataxia. At present, there is no specific therapeutic agent for treatment of the infection. No approved human vaccines are available for its prevention.
WNV has been studied in various animal models, including mice, hamsters, monkeys, and horses. The murine model is an effective in vivo experimental model to investigate viral pathogenesis and host immunity in humans. Following the initial subcutaneous or intraperitoneal inoculation in mice, WNV induces a systemic infection and eventually invades the CNS. Mice die rapidly when encephalitis develops, usually within one to two weeks. The severity and symptoms of lethal infection observed in the murine model mimic the symptoms caused by WNV infection in humans. Studies from experimental animal models, in vitro cell culture, and/or WNV patient samples have provided important insights into host immunity to WNV infection. Natural killer (NK) cells and γδ T cells are two innate lymphocytes that respond rapidly and non-specifically to viral infection. They are also known to form a unique link between innate and adaptive immunity. Moreover, the characteristics of these two cell types in adaptive immunity have been described in several disease models. In this review, we will discuss recent studies on these two unique cell types in both protective immunity and viral pathogenesis during WNV infection.
Influenza A viruses (IAV) have posed a persistent threat to global public health for centuries, through both recurrent seasonal epidemics and sporadic pandemic outbreaks. Approximately 10% of the global population is infected with an influenza virus annually, resulting in an estimated 3–5 million severe infections and 300,000–500,000 deaths. Initial signs and symptoms include acute onset of high fever, headache, cough, myalgias, and fatigue. IAV is a self-limiting infection in most healthy adults, predominantly affecting the upper respiratory tract, and typically resolves within seven days of symptom onset. However, severe infections progress to the lower respiratory tract, resulting in increased risk of respiratory failure and death. Populations at increased risk of severe influenza infection include infants, the elderly, pregnant women, and individuals with pre-existing respiratory, cardiac, neurological, or immunosuppressive conditions.
There is an increasing appreciation that a large percentage of severe or fatal influenza infections is associated with secondary bacterial infections. The contribution of bacterial infection to influenza morbidity and mortality was well documented throughout the 1918 “Spanish” influenza pandemic and in all subsequent influenza pandemics over the past century. Modern analyses of lung tissue and review of historical autopsy data from fatal 1918 influenza infections demonstrated that 95% of lethal cases were complicated by bacterial co-infection, primarily due to Streptococcus pneumoniae and Staphylococcus aureus. During the 1957 and 1968 influenza pandemics, secondary bacterial pneumonia also caused significant morbidity and mortality, with S. aureus and S. pneumoniae being the predominant bacterial pathogens. During the 2009 influenza pandemic, up to 34% of severe influenza infections managed in intensive care units and up to 55% of fatal cases were complicated by bacterial co-infections. It is estimated that approximately 65,000 influenza- and pneumonia-related deaths occur in the U.S. each year. S. aureus, including methicillin-resistant S. aureus (MRSA), is highly prevalent in severe IAV-bacterial co-infection in adults and infants.
Host and pathogen molecular mechanisms that contribute to severe influenza-bacterial infections in the lower respiratory tract are poorly understood. Excessive mucus production and impaired mucociliary clearance in response to IAV infection facilitates bacterial colonization of the lower respiratory tract, and respiratory epithelial cell barrier breakdown predisposes to bacterial invasion. Influenza infection may also enhance bacterial adhesion to cells through the incorporation of hemagglutinin into the host cell membrane, promoting bacterial cell attachment. These events, in conjunction with respiratory epithelial cell barrier breakdown, are likely critical to the development of secondary bacterial infections. Type I and type II alveolar epithelial cells, responsible for physiology gas-exchange and surfactant production, respectively, become infected by influenza viruses, and altered alveolar-capillary membrane function results in impaired oxygen exchange and lung injury. However, molecular mechanisms contributing to (1) bacterial replication, (2) bacterial virulence factor expression, and (3) host cell signaling in the context of IAV co-infection to epithelial cell barrier breakdown have not been fully elucidated. Understanding the contribution of these factors to co-infection pathogenesis may yield novel therapeutic targets for treatment of IAV and bacterial co-infection.
As the pathophysiology of severe influenza-bacterial co-infections is primarily associated with the lower respiratory tract, we sought to characterize the contributions of viral-, bacterial-, and host-mediated factors to alveolar cell dysfunction. For this analysis, we employed human adenocarcinoma A549 alveolar epithelial cells to characterize host- and pathogen contributions directly in a relevant and well-characterized alveolar epithelial cell line. Further, A549 cells have been used extensively for the analysis of host responses to influenza virus infection. We studied (1) the impact of IAV-infection on MRSA replication kinetics in A549 cell culture, (2) the host cell response to IAV, MRSA, or co-infection by analyzing temporal intracellular kinome responses, (3) the modulation of MRSA virulence factors related to adhesion and invasion in the presence or absence of IAV co-infection by RT-qPCR, and (4) alveolar epithelial barrier function and integrity during IAV, MRSA, or co-infection using electric cell-substrate impedance sensing (ECIS).
Group C viruses are antigenically characterized into the genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales. This name is historically based on their serological characteristics, which makes them distinct from members of group A (Alphaviruses genus of the family Togaviridae) and group B (Flavivirus genus of the family Flaviviridae) antigenic groups. Currently, the group C serogroup is composed of 15 distinct viruses isolated from humans, wild animals (mainly rodents, monkeys, marsupials, and bats), and mosquitoes. These viruses are present in tropical and subtropical areas of the Americas, including the United States, Mexico, Panama, Honduras, Guatemala, Trinidad, Brazil, Peru, Ecuador, Venezuela, and French Guiana [1, 3, 4].
Clinically, the human infections caused by Group C viruses are asymptomatic or characterized by unspecific febrile illness [1, 5, 6]. The percentage of asymptomatic Group C viruses infections are unknown, and apparently, the prevalence of antibodies against Group C viruses is directly related to people living nearby or maintaining close contact to forest or ecological niches in tropical areas.
The genomes of members of Group C viruses presents the typical organization of other orthobunyaviruses, which are a tri-segmented negative-sense RNA named small (SRNA), medium (MRNA) and large (LRNA) segments. The SRNA encodes a nucleocapsid protein (N protein) and a non-structural protein (NSs), while the MRNA encodes a polyprotein precursor that after a post-cleavage process gives rise to two envelope glycoproteins (Gc and Gn) and a non-structural protein (NSm). LRNA segment encodes a large RNA-dependent RNA polymerase (RdRp). So far, previous studies have described the genomic characteristics S and M RNA segments of members of Group C viruses, but many sequences generated by Sanger sequencing approach were divergent, despite using the same strains of group C viruses [1, 7–9]. Therefore, in this study, we combined high-throughput sequencing (HTS), rapid amplification of cDNA ends (RACE), and comprehensive phylogenetic analysis of complete coding sequences of Apeu virus (APEUV) strain BeAn848, Itaqui virus (ITQV) strain BeAn12797, and Nepuyo virus (NEPV) strain BeAn10709 and re-sequencing of Caraparu virus (CARV) strain BeAn3994, Madrid virus (MADV) strain BT4075, Murucutu virus (MURV) strain BeAn974, Oriboca virus (ORIV) strain BeAn17, Marituba virus (MTBV) strain BeAn15.
This was a clinical observational study of convalescent SeM antibody titers in a strangles outbreak with a high rate of complications. All samples were obtained with informed client consent. Approximately 8 weeks after initial diagnosis of infection with S. equi, serum was collected via jugular venipuncture on all 48 horses on the farm. S. equi M protein antibody titers were measured via ELISA for each horse at Equine Diagnostic Solutions LLC in Lexington, KY. At 12 weeks after initial diagnosis, serum was collected for repeat SeM antibody titers on select horses (n = 18). At 28 weeks after initial diagnosis, serum was once again collected for measurement of SeM antibody titers (n = 36). Physical examinations were performed at all time points to determine if horses were displaying any signs of disease.
Data on each horse on the property were collected from the initial diagnosis through follow‐up to removal from quarantine. Data collected included signalment, clinical signs displayed (or absence of clinical signs), nasopharyngeal lavage or guttural pouch (GP) endoscopy and lavage results for S. equi culture and polymerase chain reaction (PCR), evidence of complications, vaccination status, and survival. Affected horses were categorized according to their clinical signs of disease into 4 categories: no disease, uncomplicated case, persistent GP infection, or complicated case. No disease was defined as no clinical evidence of S. equi infection. An uncomplicated case was defined as clinical signs of 1 or more of fever, inappetance, purulent nasal discharge, and submandibular or retropharyngeal lymphadenopathy or drainage. A persistent GP infection was defined as GP infection (positive nasopharyngeal or GP lavage S. equi culture or PCR) lasting >40 days.4 A complicated case of strangles was defined as any sequelae or atypical case including signs of immune‐mediated purpura hemorrhagica, metastatic abscess formation (abscesses remote from lymph nodes of the head), secondary infections, or dysphagia. Horses with evidence of persistent GP infection and complications were categorized dually, but no horse with uncomplicated strangles had persistent GP infection.
The frequency of titers ≥1:12 800 in different disease categories was determined. Median SeM antibody titer for each category as well as for vaccination status was determined. A t test was used to analyze the difference in titer level for vaccination status. Correlations between disease category and SeM antibody titer level were analyzed by a Pearson's test. Sensitivities and specificities with 95% CI (confidence interval) for SeM antibody titers ≥1:12 800 or ≥1:6400 detecting complications or persistent GP infection were calculated. The mean reciprocal antibody titer for each time point was calculated, and regression was calculated to determine antibody decay over time. Values of P < .05 were considered statistically significant.
Members of the Coronaviridae family, OC43, 229E, NL63 and HKU1, have been associated with self-limiting respiratory tract infections in human. On the other hand, severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, cause severe respiratory disease in human. Most animal models have been established for the study of SARS-CoV and MERS-CoV which are less relevant to asthma.
HBoV was first isolated in 2005 and has been detected in both the respiratory tract and gastrointestinal tract. HBoVs belong to the family Parvoviridae, which consists of a group of small non-enveloped single-stranded DNA viruses. HBoV has been found alone in patients with respiratory complaints but more often, it is found in combination with other common respiratory viruses such as HRV and RSV. Though the presence of HBoV has been associated with clinical manifestations including rhinorrhea, pneumonia, bronchiolitis, acute wheezing and asthma exacerbation, HBoV pathogenicity remains to be fully clarified mainly due to the lack of animal models. The first trial of HBoV infection in ferret lung has recently been performed.