Immune Response to Chikungunya Virus: Sex as a Biological Variable and Implications for Natural Delivery via the Mosquito.

Chikungunya virus (CHIKV) is a mosquito-borne virus with significant public health implications around the world. Climate change, as well as rapid urbanization, threatens to expand the population range of Aedes vector mosquitoes globally, increasing CHIKV cases worldwide in return. Epidemiological data suggests a sex-dependent response to CHIKV infection. In this review, we draw attention to the importance of studying sex as a biological variable by introducing epidemiological studies from previous CHIKV outbreaks. While the female sex appears to be a risk factor for chronic CHIKV disease, the male sex has recently been suggested as a risk factor for CHIKV-associated death; however, the underlying mechanisms for this phenotype are unknown. Additionally, we emphasize the importance of including mosquito salivary components when studying the immune response to CHIKV. As with other vector-transmitted pathogens, CHIKV has evolved to use these salivary components to replicate more extensively in mammalian hosts; however, the response to natural transmission of CHIKV has not been fully elucidated.

Tick-Borne Pathogens Associated with Medically Important Ticks in Alabama: A Four-Year Survey.

Background: Tick-borne diseases (TBDs) represent a significant threat to human health in the United States. Based on reported cases of notifiable TBDs to the Centers for Disease Control and Prevention (CDC) the state of Alabama is no exception, yet previously there has been no active surveillance program in place to comprehensively assess the presence and prevalence of tick vectors and their associated TBD pathogens in Alabama. Here we summarize initial findings from a 4-year survey to address this unmet need. Materials and Methods: Beginning in 2018 and proceeding through 2021, ticks were collected throughout the state of Alabama and pooled before being screened for a panel of TBD pathogens known to circulate in the United States. Results: Consistent with previously reported cases, TBD pathogens associated with anaplasmosis, babesiosis, ehrlichiosis, and spotted fever rickettsiosis were detected in ticks of Alabama. Causative agents for tularemia and Lyme disease were not detected despite previously reported human disease cases. There was also no evidence of Heartland virus despite recent reports of the virus being detected in ticks in Northwestern counties. Conclusions: While these results serve to provide some insights into TBD pathogens associated with ticks in Alabama, they also raise many questions that highlight the need for additional studies and continued surveillance to fully understand the TBD threat to human health in Alabama.

Statewide survey of medically important ticks on white-tailed deer, Odocoileus virginianus Zimmerman, in Alabama, U.S.A.

A statewide survey of the tick fauna found on deer, using harvested deer heads as the sample unit, was conducted during the Alabama hunting seasons of 2019-2020 and 2020-2021. Four species of ticks: Ixodes scapularis (n= 936, % of catch 69.1%), Amblyomma americanum (315, 23.2%), Dermacentor albipictus (97, 7.1%), and Amblyomma maculatum (6, 0.4%) were taken from 151 deer heads harvested from 21 deer processing centers (n = 4-17 heads/processor). A total of 87.7% (prevalence) of deer heads had one or more ticks. We used two standard numerical descriptors of tick numbers, abundance, and relative abundance. No significant stepwise regressions (p > 0.05) were found between tick abundance (all ticks, I. scapularis, A. americanum) and the predictor variables of latitude, deer density, season, and year. In addition, the correlation between the abundance of I. scapularis and A. americanum was not significant (p > 0.5). In contrast, the relative abundance of both I. scapularis and A. americanum showed a significant (P < 0.05) relationship with latitude, with the relative abundance of I. scapularis increasing on deer with increased latitude and A. americanum showing the opposite pattern.

Ticks of Alabama: the fauna and spatial distribution of medically important species across the state.

The last statewide survey of hard ticks in Alabama was in 1972. To address this deficit, we examined the distribution of the medically important species across the state, Ixodes scapularis (Say), Dermacentor variabilis (Say), Amblyomma americanum (L.), and A. maculatum (Koch), between April, 2018 and February, 2021. Collections primarily involved dragging (April to July) and examination of harvested deer (November to February). A total of 2,927 ticks was collected from 110 sites; three species, I. scapularis, A. americanum, and D. variabilis, represented 91.70% of all ticks collected. Amblyomma americanum and D. variabilis were the most common species encountered in drags; I. scapularis dominated deer collections. Dermacentor variabilis was never found on deer, whereas D. albipictus was only found on deer. Stepwise regression (AIC) of drag data was linked to several site variables. Results suggest a linear response along a south (low abundance) to north (high abundance) gradient, in addition to increased abundance at sites with lower temperatures and greater precipitation and canopy cover.

Real-time tracking of bioluminescent influenza A virus infection in mice.

Despite the availability of vaccines and antiviral therapies, seasonal influenza infections cause 400,000 human deaths on average per year. Low vaccine coverage and the occurrence of drug-resistant viral strains highlight the need for new and improved countermeasures. While influenza A virus (IAV) engineered to express a reporter gene may serve as a valuable tool for real-time tracking of viral infection, reporter gene insertion into IAV typically attenuates viral pathogenicity, hindering its application to research. Here, we demonstrate that lethal or even sublethal doses of bioluminescent IAV carrying the NanoLuc gene in the C-terminus of PB2 can be tracked in real-time in live mice without compromising pathogenicity. Real-time tracking of this bioluminescent IAV enables spatiotemporal viral replication tracking in animals that will facilitate the development of countermeasures by enhancing the interpretation of clinical signs and prognosis while also allowing less animal usage.

Evaluation of DNA-Launched Virus-Like Particle Vaccines in an Immune Competent Mouse Model of Chikungunya Virus Infection.

Chikungunya virus (CHIKV) infection can result in chronic and debilitating arthralgia affecting humans in tropical and subtropical regions around the world, yet there are no licensed vaccines to prevent infection. DNA launched virus like particle (VLP) vaccines represent a potentially safer alternative to traditional live-attenuated vaccines; however, fully characterized immunocompetent mouse models which appropriately include both male and female animals for preclinical evaluation of these, and other, vaccine platforms are lacking. Utilizing virus stocks engineered to express mutations reported to enhance CHIKV virulence in mice, infection of male and female immunocompetent mice was evaluated, and the resulting model utilized to assess the efficacy of candidate DNA launched CHIKV VLP vaccines. Results demonstrate the potential utility of DNA launched VLP vaccines in comparison to a live attenuated CHIKV vaccine and identify gender differences in viral RNA loads that impact interpretation of vaccine efficacy and may have important implications for future CHIKV vaccine development.

AR12 (OSU-03012) suppresses GRP78 expression and inhibits SARS-CoV-2 replication.

AR12 is a derivative of celecoxib which no-longer acts against COX2 but instead inhibits the ATPase activity of multiple chaperone proteins, in particular GRP78. GRP78 acts as a sensor of endoplasmic reticulum stress and is an essential chaperone required for the life cycle of all mammalian viruses. We and others previously demonstrated in vitro and in vivo that AR12 increases autophagosome formation and autophagic flux, enhances virus protein degradation, preventing virus reproduction, and prolonging the survival of infected animals. In this report, we determined whether AR12 could act against SARS-CoV-2. In a dose-dependent fashion AR12 inhibited SARS-CoV-2 spike protein expression in transfected or infected cells. AR12 suppressed the production of infectious virions via autophagosome formation, which was also associated with degradation of GRP78. After AR12 exposure, the colocalization of GRP78 with spike protein was reduced. Knock down of eIF2α prevented AR12-induced spike degradation and knock down of Beclin1 or ATG5 caused the spike protein to localize in LAMP2+ vesicles without apparent degradation. HCT116 cells expressing ATG16L1 T300, found in the majority of persons of non-European descent, particularly from Africa, expressed greater amounts of GRP78 and SARS-CoV-2 receptor angiotensin converting enzyme 2 compared to ATG16L1 A300, predominantly found in Europeans, suggestive that ATG16L1 T300 expression may be associated with a greater ability to be infected and to reproduce SARS-CoV-2. In conclusion, our findings demonstrate that AR12 represents a clinically relevant anti-viral drug for the treatment of SARS-CoV-2.

Comparative pathology study of Venezuelan, eastern, and western equine encephalitis viruses in non-human primates.

Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV) are mosquito-borne viruses in the Americas that cause central nervous system (CNS) disease in humans and equids. In this study, we directly characterized the pathogenesis of VEEV, EEEV, and WEEV in cynomolgus macaques following subcutaneous exposure because this route more closely mimics natural infection via mosquito transmission or by an accidental needle stick. Our results highlight how EEEV is significantly more pathogenic compared to VEEV similarly to what is observed in humans. Interestingly, EEEV appears to be just as neuropathogenic by subcutaneous exposure as it was in previously completed aerosol exposure studies. In contrast, subcutaneous exposure of cynomolgus macaques with WEEV caused limited disease and is contradictory to what has been reported for aerosol exposure. Several differences in viremia, hematology, or tissue tropism were noted when animals were exposed subcutaneously compared to prior aerosol exposure studies. This study provides a more complete picture of the pathogenesis of the encephalitic alphaviruses and highlights how further defining the neuropathology of these viruses could have important implications for the development of medical countermeasures for the neurovirulent alphaviruses.

Single dose of DPX-rPA, an enhanced-delivery anthrax vaccine formulation, protects against a lethal Bacillus anthracis spore inhalation challenge.

Anthrax is a serious biological threat caused by pulmonary exposure to aerosolized spores of Bacillus anthracis. Biothrax® (anthrax vaccine adsorbed (AVA)) is the only Food and Drug Administration-licensed vaccine and requires five administrations over 12 months with annual boosting to maintain pre-exposure prophylaxis. Here we report the evaluation of a single intramuscular injection of recombinant B. anthracis-protective antigen (rPA) formulated in the DPX delivery platform. Immune responses were compared to an alum-based formulation in mice and rabbits. Serological analysis of anti-rPA immunoglobulin G and toxin neutralization activity demonstrated higher responses induced by DPX-rPA when compared to rPA in alum. DPX-rPA was compared to AVA in rabbits and non-human primates (NHPs). In both species, DPX-rPA generated responses after a single immunization, whereas AVA required two immunizations. In rabbits, single injection of DPX-rPA or two injections of AVA conferred 100% protection from anthrax challenge. In NHPs, single-dose DPX-rPA was 100% protective against challenge, whereas one animal in the two-dose AVA group and all saline administered animals succumbed to infection. DPX-rPA was minimally reactogenic in all species tested. These data indicate that DPX-rPA may offer improvement over AVA by reducing the doses needed for protective immune responses and is a promising candidate as a new-generation anthrax vaccine.

Comparative Pathogenesis of Asian and African-Lineage Zika Virus in Indian Rhesus Macaque's and Development of a Non-Human Primate Model Suitable for the Evaluation of New Drugs and Vaccines.

The establishment of a well characterized non-human primate model of Zika virus (ZIKV) infection is critical for the development of medical interventions. In this study, challenging Indian rhesus macaques (IRMs) with ZIKV strains of the Asian lineage resulted in dose-dependent peak viral loads between days 2 and 5 post infection and a robust immune response which protected the animals from homologous and heterologous re-challenge. In contrast, viremia in IRMs challenged with an African lineage strain was below the assay’s lower limit of quantitation, and the immune response was insufficient to protect from re-challenge. These results corroborate previous observations but are contrary to reports using other African strains, obviating the need for additional studies to elucidate the variables contributing to the disparities. Nonetheless, the utility of an Asian lineage ZIKV IRM model for countermeasure development was verified by vaccinating animals with a formalin inactivated reference vaccine and demonstrating sterilizing immunity against a subsequent subcutaneous challenge.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

A sensitive virus yield assay for evaluation of Antivirals against Zika Virus.

Despite the rapid spread of Zika virus (ZIKV) infection and associated neurological complications in the America's, prophylactic or therapeutic countermeasures are not currently available. This is mostly due to the fact that until recently there was no presumed need for medical intervention since there was no association between ZIKV infection and significant human morbidity. Consequently, there are currently no tools due mostly to the lack of sensitive cell based assays amenable for identification of ZIKV inhibitors. To address this unmet need we have developed a cell based virus yield assay suitable for testing antivirals against Zika virus. Using bioinformatics, several isolates of ZIKV from the Americas, Africa, and Asia were analyzed for sequence similarity. The alignment data were then used to design primers targeting a ZIKV genomic region that was highly conserved among all the ZIKV isolates. Subsequently, primers were used in a sensitive, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay to detect ZIKV RNA. The qRT-PCR assay was found to be highly sensitive (lower limit of detection between-10-100 copies) and reproducible. Evaluation of the primers and probes used for ZIKV against another flavivirus (Dengue virus) demonstrated specificity of detection. To evaluate potential of qRT-PCR assay as an antiviral screening tool against ZIKV, Vero cells pretreated with Type I Interferons (IFN α) were infected with virus, followed by measurement of ZIKV RNA found in the cell culture supernatants using qRT-PCR assay. Dose-dependent antiviral activity of Type I Interferons and mycophenolic acid (MPA) against Zika virus in this cell culture system was confirmed using qRT-PCR. Due to reproducible assay performance, qPCR associated higher sensitivity and short duration of the assay time, this novel cell based assay will be very useful for confirming the activity of antivirals against ZIKV.

Development of a Zika Virus Infection Model in Cynomolgus Macaques.

Limited availability of Indian rhesus macaques (IRM) is a bottleneck to study Zika virus (ZIKV) pathogenesis and evaluation of appropriate control measures in non-human primates. To address these issues, we report here the Mauritian cynomolgus macaque (MCM) model for ZIKV infection. In brief, six MCMs (seronegative for Dengue and ZIKV) were subdivided into three cohorts with a male and female each and challenged with different doses of Asian [PRVABC59 (Puerto Rico) or FSS13025 (Cambodia)] or African (IBH30656) lineage ZIKV isolates. Clinical signs were monitored; and biological fluids (serum, saliva, and urine) and tissues (testes and brain) were assessed for viral load by quantitative reverse transcription polymerase chain reaction and neutralizing antibodies (Nab) by 50% Plaque Reduction Neutralization Test (PRNT50) at various times post-infection (p.i). PRVABC59 induced viremia detectable up to day 10, with peak viral load at 2-3 days p.i. An intermittent viremia spike was observed on day 30 with titers reaching 2.5 × 103 genomes/mL. Moderate viral load was observed in testes, urine and saliva. In contrast, FSS13025 induced viremia lasting only up to 6 days and detectable viral loads in testes but not in urine and saliva. Recurrent viremia was detected but at lower titers compare to PRVABC59. Challenge with either PRVABC59 or FSS13025 resulted in 100% seroconversion; with mean PRNT50 titers ranging from 597 to 5179. IBH30656 failed to establish infection in MCM suggesting that MCM are susceptible to infection with ZIKV isolates of the Asian lineage but not from Africa. Due to the similarity of biphasic viremia and Nab responses between MCM and IRM models, MCM could be a suitable alternative for evaluation of ZIKV vaccine and therapeutic candidates.

Combined alphavirus replicon particle vaccine induces durable and cross-protective immune responses against equine encephalitis viruses.

Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV, or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV-IAB virus, Trinidad donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American variety of EEEV. Both the WEEV replicon and combination V/W/E vaccination, however, elicited poor neutralizing antibodies to WEEV in macaques, and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal. Importance: Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV). Normally transmitted by mosquitoes, these viruses can cause disease when inhaled, so there is concern that these viruses could be used as biological weapons. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis expressing the surface proteins of all three viruses. In this report we demonstrate in both mice and macaques that this combined vaccine is safe, generates a strong immune response, and protects against aerosol challenge with the viruses that cause Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis.

Differential accumulation of genetic and phenotypic changes in Venezuelan equine encephalitis virus and Japanese encephalitis virus following passage in vitro and in vivo.

The requirement to replicate in both vertebrate and invertebrate hosts is thought to limit the introduction of genetic changes into the genome of arboviruses. Serial passage under laboratory conditions will overcome this limitation allowing for genetic changes to be introduced and affecting the virulence of the virus for animals. In the studies detailed here, the consequence of removing the restriction of alternate replication was demonstrated to be different depending on the virus. Passing Venezuelan equine encephalitis virus in tissue culture cells, eggs or mice resulted in up to 11 nucleotide or amino acid changes but no significant change in the virulence of the virus for mice. Passing Japanese encephalitis virus (JEV) under the identical conditions resulted in as many as 22 nucleotide or amino acid changes that often resulted in improved survival probabilities. For JEV, most genetic changes along with the attenuated phenotype were selected within 5 passes.

Gas-permeable ethylene bags for the small scale cultivation of highly pathogenic avian influenza H5N1 and other viruses in embryonated chicken eggs.

BACKGROUND: Embryonated chicken eggs (ECE) are sometimes used for the primary isolation or passage of influenza viruses, other viruses, and certain bacteria. For small-scale experiments with pathogens that must be studied in biosafety level three (BSL3) facilities, inoculated ECE are sometimes manipulated and maintained in small egg incubators within a biosafety cabinet (BSC). To simplify the clean up and decontamination of an egg incubator in case of egg breakage, we explored whether ethylene breather bags could be used to encase ECE inoculated with pathogens. This concept was tested by determining embryo survival and examining virus yields in bagged ECE. RESULTS: Virus yields acceptable for many applications were attained when influenza-, alpha-, flavi-, canine distemper-, and mousepox viruses were propagated in ECE sealed within ethylene breather bags. CONCLUSIONS: For many small-scale applications, ethylene breather bags can be used to encase ECE inoculated with various viruses.

Sterile protection against Plasmodium knowlesi in rhesus monkeys from a malaria vaccine: comparison of heterologous prime boost strategies.

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost.

Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

BACKGROUND: The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. CONCLUSIONS/SIGNIFICANCE: We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

A heterologous DNA prime-Venezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus challenge in cynomolgus macaques.

A candidate vaccine (D1ME-VRP) expressing dengue virus type 1 premembrane and envelope proteins in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) system was constructed and tested in conjunction with a plasmid DNA vaccine (D1ME-DNA) expressing identical dengue virus sequences. Cynomolgus macaques were vaccinated with three doses of DNA (DDD), three doses of VRP (VVV group), or a heterologous DNA prime-VRP boost regimen (DDV) using two doses of DNA vaccine and a third dose of VRP vaccine. Four weeks after the final immunization, the DDV group produced the highest dengue virus type 1-specific immunoglobulin G antibody responses and virus-neutralizing antibody titers. Moderate T-cell responses were demonstrated only in DDD- and DDV-vaccinated animals. When vaccinated animals were challenged with live virus, all vaccination regimens showed significant protection from viremia. DDV-immunized animals were completely protected from viremia (mean time of viremia = 0 days), whereas DDD- and VVV-vaccinated animals had mean times of viremia of 0.66 and 0.75 day, respectively, compared to 6.33 days for the control group of animals.

Uncommon respiratory pathogens.

PURPOSE OF REVIEW: In many cases, the specific pathogen responsible for a respiratory infection is not identified and can lead to improper medical treatment, increased duration of illness, and possibly contributes to the development of antibiotic resistance. Molecular-based diagnostic methodologies have significantly improved our ability to identify common respiratory pathogens; these techniques are not useful, however, when a novel pathogen is responsible for the infection and clinicians must rely on differential diagnosis for the treatment of patients. RECENT FINDINGS: New pathogens previously not associated with human infections have been identified in the past few years. In addition, new strains of bacteria and viruses have emerged as the causative agents of pneumonia and acute respiratory distress. Protozoans and saprophytic fungi, which are not normally associated with respiratory infection, have also emerged as respiratory pathogens particularly in individuals with AIDS or in those who are otherwise immunocompromised. SUMMARY: This review discusses the recent literature on newly described respiratory pathogens as well as opportunistic pathogens that can infect the respiratory system of immunocompromised individuals. The studies referenced here reveal the need for expanded laboratory tests and highly trained microbiologists in clinical laboratories worldwide.