IFITM Proteins That Restrict the Early Stages of Respiratory Virus Infection Do Not Influence Late-Stage Replication.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.

Frequent lower respiratory tract disease in hematological patients with parainfluenza virus type 3 infection.

Human parainfluenza virus type 3 (HPIV-3) may cause lower respiratory tract infection disease (LRTI-D) after hematopoietic stem cell transplantation (HSCT). Most previous have studies focused on recipients of HSCT whereas data on characteristics and outcomes in patients with hematological malignancies (HMs) compared to non-hematological patients are limited. The prognostic value of viral load in respiratory specimens remains elusive. In a 2-year retrospective study, we determined the frequencies of LRTI-D in HM, HSCT, and in non-hematological patients, and HPIV-3 levels in respiratory tract secretions. Among 98 patients with HPIV-3 infection, including 31 HSCT and 40 HM, 36 had a diagnosis of LRTI-D. LRTI-D was significantly more frequent in patients with HM or HSCT (n = 32, 45.1%) than in non-hematological patients (n = 4, 14.8%) (p = 0.006). The median HPIV-3 loads were high in upper respiratory tract secretions regardless of the presence or absence of LRTI-D (8.3 log10 vs. 7.6 log10 TCID50 /106 cells). HPIV-3 loads in respiratory tract samples in HM were not significantly higher than those found in HSCT but significantly higher than in non-hematological patients (p = 0.007). In conclusion, LRTI-D was frequent in HM patients who were diagnosed with HPIV-3 infection.

Caprine parainfluenza virus type 3 N protein promotes viral replication via inducing apoptosis.

Caprine parainfluenza virus type 3 (CPIV3) is one of the most important viral respiratory pathogens of goat. Accumulating evidence demonstrates that apoptosis is a cellular mechanism for the host response to pathogens, and it participates in regulating viral replication. However, there is little study on CPIV3-induced host cells apoptosis. In this study, primary goat tracheal epithelial (GTE) cells were established as a cellular model that is permissive to CPIV3 infection. Then, we showed that CPIV3 infection induced apoptosis in GTE cells, as determined by morphological changes, flow cytometry and TUNEL assay. Moreover, Caspase activity and the expression of pro-apoptotic genes further suggested that CPIV3 induced apoptosis by activating both the intrinsic and extrinsic pathways. Mechanistically, the ability of CPIV3 to induce apoptosis was activated by N protein, and the viral protein increased CPIV3 replication through effecting apoptosis. Overall, our findings showed that GTE cells that will enable further analysis of CPIV3 infection and offers novel insights into the mechanisms of CPIV3-induced apoptosis in host cells.

Protective antibodies against human parainfluenza virus type 3 infection.

Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

Dynamics of nosocomial parainfluenza virus type 3 and influenza virus infections at a large German University Hospital between 2012 and 2019.

Nosocomial virus infections cause significant morbidity and mortality. Besides influenza viruses, the disease burden of parainfluenza virus type 3 (PIV-3) is comparatively high among hospitalized patients and severe disease courses can occur. PIV-3 showed the highest rates of nosocomial infections of a panel of respiratory viruses. Therefore, a retrospective observational study was conducted among patients with either PIV-3 or influenza viruses, which served as reference pathogen. The aim was to compare the seasonal dynamics and clinical characteristics of nosocomial infections with these highly transmittable viruses. Nosocomial infection occurred in 15.8% (n = 177) of all influenza cases, mainly in the first half of a season. About 24.3% (n = 104) of the PIV-3 cases were nosocomial and occurred mainly in the second half of a season. Both nosocomial rates of influenza and nosocomial rates of PIV-3 varied between the seasons. Community acquired and nosocomial cases differed in underlying medical conditions and immunosuppression. Knowledge of the baseline rates of nosocomial infections could contribute to the implementation of appropriate infection control measures.

T-Cell Therapeutics Targeting Human Parainfluenza Virus 3 Are Broadly Epitope Specific and Are Cross Reactive With Human Parainfluenza Virus 1.

Human Parainfluenza Virus-3 (HPIV3) causes severe respiratory illness in immunocompromised patients and lacks approved anti-viral therapies. A phase I study of adoptively transferred virus-specific T-cells (VSTs) targeting HPIV3 following bone marrow transplantation is underway (NCT03180216). We sought to identify immunodominant epitopes within HPIV3 Matrix protein and their cross-reactivity against related viral proteins. VSTs were generated from peripheral blood of healthy donors by ex-vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 matrix protein. Epitope mapping was performed using IFN-γ ELIspot with combinatorial peptide pools. Flow cytometry was used to characterize products with intracellular cytokine staining. In 10 VST products tested, we discovered 12 novel immunodominant epitopes. All products recognized an epitope at the C-terminus. On IFN-γ ELISpot, individual peptides eliciting activity demonstrated mean IFN-γ spot forming units per well (SFU)/1x105 cells of 115.5 (range 24.5-247.5). VST products were polyfunctional, releasing IFN-γ and TNF-α in response to identified epitopes, which were primarily HLA Class II restricted. Peptides from Human Parainfluenza Virus-1 corresponding to the HPIV3 epitopes showed cross-reactivity for HPIV1 in 11 of 12 tested epitopes (mean cross reactivity index: 1.19). Characterization of HPIV3 epitopes may enable development of third-party VSTs to treat immune suppressed patients with HPIV infection.

PI4KB on Inclusion Bodies Formed by ER Membrane Remodeling Facilitates Replication of Human Parainfluenza Virus Type 3.

Many positive-strand RNA viruses remodel the endomembrane to form specialized replication organelles. However, knowledge regarding whether negative-strand RNA viruses take advantage of intracellular membranes for replication is limited. Here we show that a negative-strand RNA virus, human parainfluenza virus type 3 (HPIV3), remodels the endoplasmic reticulum (ER) membrane to form inclusion bodies (IBs), whereby the phosphoprotein (P) of HPIV3 recruits phosphatidylinositol 4-kinase beta (PI4KB) to IBs to generate PI4P, creating a PI4P-enriched microenvironment to promote HPIV3 replication. In addition, we find that human respiratory syncytial virus (HRSV) also takes advantage of the ER to form IBs and that these IBs are also enriched with PI4P. The nucleoprotein of HRSV recruits PI4KB to IBs. These results suggest that paramyxoviruses also exploit the host endomembrane to form IBs and that PI4KB is recruited by viral proteins to enrich IBs with PI4P to facilitate viral replication.

The novel caprine parainfluenza virus type 3 showed pathogenicity in Guinea pigs.

Caprine parainfluenza virus type 3 (CPIV3) is one of the important viral respiratory tract agents in goats. The pathogenicity of CPIV3 has been examined in goats but it has not been explored in other laboratory animals. In the present study, an experimental infection of guinea pigs with CPIV3 was performed. The virus-inoculated guinea pigs displayed clinical signs related to the respiratory disease at 2-12 days post inoculation (dpi). Five infected guinea pigs died during 2 and 7 dpi. Apparent gross pneumonic lesions including consolidation and congestion in one or more lung lobes were observed in necropsied and dead animals. Histo-pathological changes in lungs including expansions of the alveolar interstitium, congestion, macrophage infiltration and compensatory emphysema were also observed. Virus was detectable at 2-10 dpi, 2-10 dpi and 2-7 dpi, as detected by virus isolation, real-time RT-PCR and immunohistochemistry staining, respectively. Viremia was also confirmed after CPIV3 infection during 3-7 dpi. The severe pathological lesions and highest viral load were observed before 7 dpi. Viral specific hemagglutination inhibition and neutralizing antibodies were produced from 7 dpi and 10 dpi, respectively, which related to the clearance of virus. The results present here indicated that guinea pig could be an ideal laboratory animal model for CPIV3 studies in the future.

Authentic Modeling of Human Respiratory Virus Infection in Human Pluripotent Stem Cell-Derived Lung Organoids.

Infectious viruses so precisely fit their hosts that the study of natural viral infection depends on host-specific mechanisms that affect viral infection. For human parainfluenza virus 3, a prevalent cause of lower respiratory tract disease in infants, circulating human viruses are genetically different from viruses grown in standard laboratory conditions; the surface glycoproteins that mediate host cell entry on circulating viruses are suited to the environment of the human lung and differ from those of viruses grown in cultured cells. Polarized human airway epithelium cultures have been used to represent the large, proximal airways of mature adult airways. Here we modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids derived from human pluripotent stem cells contain mesoderm and pulmonary endoderm and develop into branching airway and alveolar structures. Whole-genome sequencing analysis of parainfluenza viruses replicating in the organoids showed maintenance of nucleotide identity, suggesting that no selective pressure is exerted on the virus in this tissue. Infection with parainfluenza virus led to viral shedding without morphological changes, while respiratory syncytial virus infection induced detachment and shedding of infected cells into the lung organoid lumens, reminiscent of parainfluenza and respiratory syncytial virus in human infant lungs. Measles virus infection, in contrast, induced syncytium formation. These human stem cell-derived lung organoids may serve as an authentic model for respiratory viral pathogenesis in the developing or infant lung, recapitulating respiratory viral infection in the host.IMPORTANCE Respiratory viruses are among the first pathogens encountered by young children, and the significant impact of these viral infections on the developing lung is poorly understood. Circulating viruses are suited to the environment of the human lung and are different from those of viruses grown in cultured cells. We modeled respiratory virus infections that occur in children or infect the distal lung using lung organoids that represent the entire developing infant lung. These 3D lung organoids, derived from human pluripotent stem cells, develop into branching airway and alveolar structures and provide a tissue environment that maintains the authentic viral genome. The lung organoids can be genetically engineered prior to differentiation, thereby generating tissues bearing or lacking specific features that may be relevant to viral infection, a feature that may have utility for the study of host-pathogen interaction for a range of lung pathogens.

The Matrix Protein of Human Parainfluenza Virus Type 3 Induces Mitophagy that Suppresses Interferon Responses.

Mitophagy is a form of autophagy that selectively removes damaged mitochondria. Impaired mitochondria can be tagged by the kinase PINK1, which triggers recruitment of the E3-ubiquitin ligase Parkin and subsequent mitochondrial sequestration within autophagosomes. We previously found that human parainfluenza virus type 3 (HPIV3) infection induces autophagy, but the type and mechanisms of autophagy induction remain unknown. Here, we show that matrix protein (M) of HPIV3 translocates to mitochondria and interacts with Tu translation elongation factor mitochondrial (TUFM). M-mediated mitophagy does not require the Parkin-PINK1 pathway but rather an interaction between M and the LC3 protein that mediates autophagosome formation. These interactions with both TUFM and LC3 are required for the induction of mitophagy and lead to inhibition of the type I interferon response. These results reveal that a viral protein is sufficient to induce mitophagy by bridging autophagosomes and mitochondria.

Agenesia aislada de la arteria pulmonar derecha / Isolated right pulmonary artery agenesis

No disponible

Parainfluenza 3 Infections Early After Kidney or Simultaneous Pancreas-Kidney Transplantation.

Parainfluenza virus (PIV) can cause serious infections after hematopoietic stem cell or lung transplantation. Limited data exist about PIV infections after kidney transplantation. We describe an outbreak of PIV-3 in a transplant unit. During the outbreak, 45 patients were treated on the ward for postoperative care after kidney or simultaneous pancreas-kidney (SPK) transplantation. Overall, 29 patients were tested for respiratory viruses (12 patients with respiratory symptoms, 17 asymptomatic exposed patients) from nasopharyngeal swabs using polymerase chain reaction. PIV-3 infection was confirmed in 12 patients. One patient remained asymptomatic. In others, symptoms were mostly mild upper respiratory tract symptoms and subsided within a few days with symptomatic treatment. Two patients suffered from lower respiratory tract symptoms (dyspnea, hypoxemia, pulmonary infiltrates in chest computed tomography) and required supplemental oxygen. Four of six SPK patients and eight of 39 of kidney transplant patients were infected with PIV (p = 0.04). In patients with follow-up tests, PIV-3 shedding was still detected 11-16 days after diagnosis. Despite rapid isolation of symptomatic patients, PIV-3 findings were diagnosed within 24 days, and the outbreak ceased only after closing the transplant ward temporarily. In conclusion, PIV-3 infections early after kidney or SPK transplantation were mostly mild. PIV-3 easily infected immunosuppressed transplant recipients, with prolonged viral shedding.

Parainfluenza Virus Types 1, 2, and 3 in Pediatric Patients with Acute Respiratory Infections in Beijing During 2004 to 2012.

BACKGROUND: Although human parainfluenza virus (HPIV) has been determined as an important viral cause of acute respiratory infections (ARIs) in infants and young children, data on long-term investigation are still lacking to disclose the infection pattern of HPIV in China. METHODS: Nasopharyngeal aspirates were collected from 25,773 hospitalized pediatric patients with ARIs from January 2004 through December 2012 for respiratory virus screen by direct immuno-fluorescence assay. RESULTS: Out of these specimens, 1675 (6.50%, 1675/25,773) showed HPIV positive, including 261 (1.01%, 261/25,773) for HPIV1, 28 (0.11%, 28/25,773) for HPIV2, and 1388 (5.39%, 1388/25,773) for HPIV3, 2 of the samples were positive for both HPIV1 and HPIV3, and 36 were co-detected with other viruses. The positive rates of HPIVs were higher in those younger than 3 years old. HPIV3 was detected from all age groups, predominantly from patients under 3 years of age, and the highest frequency was found in those 6 months to 1-year old (352/4077, 8.63%). HPIV3 was the dominant type in each of the years detected between May and July. HPIV1 showed a peak in every odd year, mainly in August or September. HPIV was detected most frequently from patients with upper respiratory infection (12.49%, 157/1257), followed by bronchitis (11.13%, 176/2479), asthma (9.31%, 43/462), bronchiolitis (5.91%, 150/2536), pneumonia (6.06%, 1034/17,068), and those with underlying diseases (1.0%, 15/1506). HPIV3 is the dominant type in these six disease groups referred above, especially in the asthma group. CONCLUSIONS: HPIV is one of the important viral causes of ARIs in infants and young children in Beijing based on the data from the hospitalized children covering a 9-year term. HPIV3 is the predominant type in all these years and in most of the disease groups. HPIVs with different types show different seasonality.

Terminal sialic acid linkages determine different cell infectivities of human parainfluenza virus type 1 and type 3.

Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) initiate infection by sialic acid binding. Here, we investigated sialic acid linkage specificities for binding and infection of hPIV1 and hPIV3 by using sialic acid linkage-modified cells treated with sialidases or sialyltransferases. The hPIV1 is bound to only α2,3-linked sialic acid residues, whereas hPIV3 is bound to α2,6-linked sialic acid residues in addition to α2,3-linked sialic acid residues in human red blood cells. α2,3 linkage-specific sialidase treatment of LLC-MK2 cells and A549 cells decreased the infectivity of hPIV1 but not that of hPIV3. Treatment of A549 cells with α2,3 linkage-specific sialyltransferase increased infectivities of both hPIV1 and hPIV3, whereas α2,6 linkage-specific sialyltransferase treatment increased only hPIV3 infectivity. Clinical isolates also showed similar sialic acid linkage specificities. We concluded that hPIV1 utilizes only α2,3 sialic acid linkages and that hPIV3 makes use of α2,6 sialic acid linkages in addition to α2,3 sialic acid linkages as viral receptors.

Evaluation of a novel immunogenic vaccine platform based on a genome replication-deficient Sendai vector.

We developed a novel vaccine platform based on a paramyxoviral, genome replication-deficient Sendai virus vector that can express heterologous genes inserted into the genome. To validate the novel approach in vivo, we generated a combined vaccine candidate against human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (PIV3). The present study compares two different methods of displaying heterologous antigens: (i) the RSV fusion (F) protein, encoded as a secretable version in an additional transcription unit, serves as an antigen only after being expressed in infected cells; (ii) PIV3 fusion (F) and hemagglutinin-neuraminidase (HN) genes, replacing Sendai counterparts in the vector genome, are also expressed as structural components on the surface of vaccine particles. The efficacy of this prototype vaccine was assessed in a mouse model after mucosal administration. The vaccine candidate was able to elicit specific mucosal, humoral and T cell-mediated immune responses against RSV and PIV3. However, PIV3 antigen display on the vaccine particles' surface induced higher antibody titers than the RSV antigen, being expressed only after cell infection. Consequently, this construct induced an adequate neutralizing antibody response only to PIV3. Finally, replicating virus particles were not detected in the lungs of immunized mice, confirming the genome stability and replication deficiency of this vaccine vector in vivo. Both factors can contribute substantially to the safety profile of vaccine candidates. In conclusion, this replication-deficient Sendai vector represents an efficient platform that can be used for vaccine developments against various viral pathogens.

Human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium.

Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.

Adaptation of human parainfluenza virus to airway epithelium reveals fusion properties required for growth in host tissue.

UNLABELLED: Paramyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). We have previously shown that viruses with an HN/F pair that is highly fusogenic in monolayers of immortalized cells due to mutations in HN's secondary sialic acid binding site are growth impaired in differentiated human airway epithelium (HAE) cultures and in vivo. Here we have shown that adaptation of HPIV3 to growth in the lung is determined by specific features of HN and F that are different from those required for growth in cultured immortalized cells. An HPIV3 virus bearing a mutated HN (H552Q), which is fit and fusogenic in immortalized cells but unfit for growth in the lung, evolved into a less-fusogenic but viable virus in differentiated human airway epithelium. Stepwise evolution led to a progressive decrease in efficiency of fusion activation by the HN/F pair, with a mutation in F first decreasing the activation of F by HN and a mutation in HN's secondary sialic acid binding site decreasing fusion activation further and producing a stable virus. Adaptation of HPIV3 to successful growth in HAE is determined by specific features of HN and F that lead to a less easily activated fusion mechanism. IMPORTANCE: Human parainfluenza viruses (HPIVs) are paramyxoviruses that cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide, but there are currently no vaccines or antivirals available for treatment. Enveloped viruses must fuse their membrane with the target cell membrane in order to initiate infection. Parainfluenza virus fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. The receptor-binding hemagglutinin-neuraminidase (HN), upon receptor engagement, activates the fusion protein (F) to penetrate the target cell and mediate viral entry. In this study, we show that the precise balance of fusion activation properties of these two glycoproteins during entry is key for infection. In clinically relevant tissues, viruses evolve to acquire a set of fusion features that provide key clues about requirements for infection in human beings.

Mutations within the human parainfluenza virus type 3 (HPIV 3) C protein affect viral replication and host interferon induction.

Human parainfluenza virus type 3 (HPIV 3) encodes a multifunctional C protein that is capable of inhibiting viral replication and counteracting the host interferon (IFN) signaling pathway. We recently demonstrated that the C protein is phosphorylated both in vitro and in vivo and mutations within the phosphorylation sites exhibit differential inhibitory activities in vitro. In this study, we report for the first time the successful recovery of mutant HPIV 3 viruses containing mutations within the C protein. Three mutant viruses, Cm-1, Cm-3 and Cm-4, harboring individual mutations of S7, S47T48 and S81 residues, respectively, were examined for their replication profiles and their ability to abrogate host IFN induction. Viral transcription was similar for all viruses; however Cm-3 displayed a relatively higher replication. Infection of cells with Cm-1 and Cm-3 led to the activation of IFN regulatory transcription factor 3 (IRF-3) and subsequent increase in IFN-ß mRNA levels as determined by immunofluorescence assay and RT-PCR analyses, respectively. Moreover, Cm-3 was able to partially resist the interferon induced antiviral state in Vero cells. Taken together, these results suggest that mutations within the C protein differentially affect viral replication and host interferon induction.

Severe pneumonia caused by combined infection with Pneumocystis jiroveci, parainfluenza virus type 3, cytomegalovirus, and Aspergillus fumigatus in a patient with Stevens-Johnson syndrome/toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe adverse cutaneous reactions to drugs. We report here the first case of severe pneumonia caused by an unusual combined infection with Pneumocystis carinii (jiroveci), parainfluenza virus type 3, cytomegalovirus and Aspergillus fumigatus in a 63-year-old female patient with allopurinol-induced SJS/TEN overlap syndrome. Following treatment with high-dose systemic corticosteroids and intravenous immunoglobulin for SJS/TEN, her mucocutaneous lesions improved and she was due to be discharged. However, 15 days after cessation of corticosteroids, she developed pneumonia. Broncho-alveolar lavage revealed that the cause of infection was Pneumocystis carinii (jiroveci), parainfluenza virus type 3, cytomegalovirus and Aspergillus. These findings indicate that patients with SJS/TEN, particularly those treated with systemic corticosteroids, may be susceptible to infection with combinations of pathological agents resulting from damage to the bronchial epithelia.