Distinct severe acute respiratory syndrome coronavirus-induced acute lung injury pathways in two different nonhuman primate species.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), caused by influenza A virus H5N1 and severe acute respiratory syndrome coronavirus (SARS-CoV), supposedly depend on activation of the oxidative-stress machinery that is coupled with innate immunity, resulting in a strong proinflammatory host response. Inflammatory cytokines, such as interleukin 1ß (IL-1ß), IL-8, and IL-6, play a major role in mediating and amplifying ALI/ARDS by stimulating chemotaxis and activation of neutrophils. To obtain further insight into the pathogenesis of SARS-CoV-associated ALI, we compared SARS-CoV infections in two different nonhuman primate species, cynomolgus macaques and African green monkeys. Viral titers in the upper and lower respiratory tract were not significantly different in SARS-CoV-infected macaques and African green monkeys. Inflammatory cytokines that play a major role in mediating and amplifying ALI/ARDS or have neutrophil chemoattractant activity, such as IL-6, IL-8, CXCL1, and CXCL2, were, however, induced only in macaques. In contrast, other proinflammatory cytokines and chemokines, including osteopontin and CCL3, were upregulated in the lungs of African green monkeys to a significantly greater extent than in macaques. Because African green monkeys developed more severe ALI than macaques, with hyaline membrane formation, some of these differentially expressed proinflammatory genes may be critically involved in development of the observed pathological changes. Induction of distinct proinflammatory genes after SARS-CoV infection in different nonhuman primate species needs to be taken into account when analyzing outcomes of intervention strategies in these species.

Exacerbated innate host response to SARS-CoV in aged non-human primates.

The emergence of viral respiratory pathogens with pandemic potential, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza A H5N1, urges the need for deciphering their pathogenesis to develop new intervention strategies. SARS-CoV infection causes acute lung injury (ALI) that may develop into life-threatening acute respiratory distress syndrome (ARDS) with advanced age correlating positively with adverse disease outcome. The molecular pathways, however, that cause virus-induced ALI/ARDS in aged individuals are ill-defined. Here, we show that SARS-CoV-infected aged macaques develop more severe pathology than young adult animals, even though viral replication levels are similar. Comprehensive genomic analyses indicate that aged macaques have a stronger host response to virus infection than young adult macaques, with an increase in differential expression of genes associated with inflammation, with NF-kappaB as central player, whereas expression of type I interferon (IFN)-beta is reduced. Therapeutic treatment of SARS-CoV-infected aged macaques with type I IFN reduces pathology and diminishes pro-inflammatory gene expression, including interleukin-8 (IL-8) levels, without affecting virus replication in the lungs. Thus, ALI in SARS-CoV-infected aged macaques developed as a result of an exacerbated innate host response. The anti-inflammatory action of type I IFN reveals a potential intervention strategy for virus-induced ALI.

Unraveling the complexities of the interferon response during SARS-CoV infection.

Viruses employ different strategies to circumvent the antiviral actions of the innate immune response. SARS coronavirus (SARS-CoV), a virus that causes severe lung damage, encodes an array of proteins able to inhibit induction and signaling of type-I interferons. However, recent studies have demonstrated that interferons are produced during SARS-CoV infection in humans and macaques. Furthermore, nuclear translocation of activated STAT1 and a range of interferon-stimulated genes could be demonstrated in the lungs of SARS-CoV-infected macaques. In line with these observations, plasmacytoid dendritic cells have been shown to produce interferons upon SARS-CoV infection in vitro. Given the pivotal role of interferons during viral infections, (differential) induction of interferons may affect the outcome of the infection. Therefore, the functional implication of interferon production during SARS-CoV infection remains to be re-investigated.

Functional genomics highlights differential induction of antiviral pathways in the lungs of SARS-CoV-infected macaques.

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. Using functional genomics, we analyzed early host responses to SARS-CoV infection in the lungs of adolescent cynomolgus macaques (Macaca fascicularis) that show lung pathology similar to that observed in human adults with SARS. Analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (IL)-6, IL-8, and IP-10, which corresponds to the host response seen in acute respiratory distress syndrome. As opposed to many in vitro experiments, SARS-CoV induced a wide range of type I interferons (IFNs) and nuclear translocation of phosphorylated signal transducer and activator of transcription 1 in the lungs of macaques. Using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. Our studies emphasize that the induction of early IFN signaling may be critical to confer protection against SARS-CoV infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of SARS.

Interferon-gamma and interleukin-4 downregulate expression of the SARS coronavirus receptor ACE2 in Vero E6 cells.

Interferons (IFNs) inhibit severe acute respiratory syndrome coronavirus (SARS-CoV) replication and might be valuable for SARS treatment. In this study, we demonstrate that treatment of Vero E6 cells with interleukin-4 (IL-4) decreased the susceptibility of these cells to SARS-CoV infection. In contrast to IFNs, IL-4 did not show antiviral activity when administered immediately after SARS-CoV infection, suggesting that IL-4 acts early during the SARS-CoV replication cycle. Indeed, binding of recombinant SARS-CoV spike protein to Vero E6 cells was diminished on cells treated with IL-4, but also on cells exposed to IFN-gamma. Consistent with these observations, IL-4 and IFN-gamma downregulated cell surface expression of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Besides diminished ACE2 cell surface expression, ACE2 mRNA levels were also decreased after treatment with these cytokines. These findings suggest that IL-4 and IFN-gamma inhibit SARS-CoV replication partly through downregulation of ACE2.

Significance of HPV tests on women with cervical smears showing ASCUS.

BACKGROUND: The relationship between the human papillomavirus (HPV) tests and the cytological findings was evaluated. METHODS: Totally, 161 women participating in the organized cytological screening in the County of Uppsala and showing atypical squamous cells of uncertain significance (ASCUS) in their cervical smears were identified during 2002 and 2003. Those women were invited for a repeated examination about 3 months later. At that occasion, a smear sample was collected and used for cytological examination and a concomitant HPV test. Oncogenic HPV was identified by polymerase chain reaction and HPV deoxyribonucleoside acid (DNA) sequence analysis in 50 cases and by Hybrid Capture II in 111 cases. RESULTS: Women with an ASCUS diagnosis showed a normal cytology and a negative HPV test in 30% of the cases, abnormal cytology and a positive HPV test in 32% of the cases; in 26% of the women, the HPV test was positive, whereas cytology was normal; and in 11% of the women, the HPV test was negative, whereas cytology was abnormal (ASCUS or more). CONCLUSION: The results show that HPV tests identify a larger group of women at risk to develop cervical cancer in comparison with cytological examination. As the HPV tests have a higher sensitivity than the cytological screening, it is suggested that an HPV test in conjunction with a diagnosis of ASCUS can be employed to more efficiently select women, to whom further follow up is recommended.

Sensitivity of HPV tests on stained vs. unstained cervical smears.

OBJECTIVE: To examine the effect of Pspanicolaou staining of cervical smears on the sensitivity of molecular biologic HPV tests. STUDY DESIGN: Two sensitive HPV tests were used, HPV DNA sequence analysis after polymerase chain reaction (PCR) amplification and the Hybrid Capture II method (HC II) (Digene Diagnostics Inc., Silver Spring, Maryland, USA). Papanicolaou-stained and unstained smears taken simultaneously were examined from 265 women readmitted for examination due to an atypical squamous cells of undetermined significance diagnosis. RESULTS: After an HPV test with the PCR method on unstained slides, 66% of the women were HPV positive, whereas the same women were HPVpositive in 54% when Papanicolaou-stained slides were analyzed. However, this difference was not statistically significant (p > 0.1). With the HC II method, 55% of unstained smears were HPV positive whereas 29% were HPV positive, when Papanicolaou-stained slides were examined. This difference was significant (p < 0.001). The same strong differences in sensitivity were observed when both the PCR and HC II methods were studied on the same Papanicolaou stained glass slides, whereas on unstained slides no significant difference was found. CONCLUSION: The results demonstrate that Papanicolaou staining of a cervical smear significantly decreases the sensitivity of an HPV test performed with the HC II method, whereas the PCR method is less affected. With the Papanicolaou method, the hematoxylin bath is followed by HCl treatment, and strong acid treatment destroys DNA.