Tropism and Innate Host Responses of the 2009 Pandemic H1N1 Influenza Virus in ex Vivo and in Vitro Cultures of Human Conjunctiva and Respiratory Tract

The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33°C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy.The recent pandemic caused by a novel H1N1 virus (H1N1pdm) arose from the reassortment of three or more viruses of swine origin, including the North American triple reassortant H3N2 and H1N2 viruses, classical swine H1N1, and European swine H1N1/H3N2 viruses.^1,2 Most patients with pandemic H1N1pdm have mild influenza-like illness, but a minority of patients develop a primary viral pneumonia, sometimes leading to acute respiratory distress syndrome and death.^3,4 Many, but not all, patients with severe disease have pregnancy, obesity, or underlying disease states such as asthma, obstructive airways disease, diabetes, and chronic cardiovascular or renal disease. The disease associated with H1N1pdm so far appears to be comparable with that of seasonal influenza and less severe than that seen in the 1918 pandemic or in zoonotic disease caused by highly pathogenic avian influenza (HPAI) H5N1. However, unlike seasonal influenza where morbidity and mortality are mainly seen in the elderly, pandemic H1N1pdm appears to spare this age-group, possibly because of the presence of cross-neutralizing antibody generated by prior repeated seasonal H1N1 infection.⁵ In California, the median age of all cases was 17 years, of hospitalized cases 26 years, and for fatal cases was 45 years.It is therefore important to understand how the pathogenesis and tissue tropism of H1N1pdm virus in humans differs from seasonal influenza viruses. However, there is so far limited information in this regard. The H1N1pdm virus does not possess the genetic motifs of virulence associated with either the HPAI H5N1 or 1918 H1N1 viruses.² In experimentally infected ferrets, macaques, and mice, H1N1pdm causes moderately more severe illness compared with seasonal influenza although being much less virulent than HPAI H5N1 or the 1918 pandemic Spanish flu virus.^6,7,8 In these animal models, H1N1pdm virus was able to infect the alveolar epithelium more readily than seasonal H1N1 virus, but whether this holds true for humans is not known.⁷ Though H1N1pdm was initially reported to have a predominantly α2-6 sialic acid (Sia) receptor binding preference⁸ similar to human seasonal influenza viruses, recent glycan array data indicates that there is binding to both “human” Sia α2-6 and “avian” Sia α2-3.⁹ H1N1pdm virus differs from seasonal influenza viruses in their ability to infect and cause illness in mice without prior adaptation. As the mouse respiratory tract has a predominance of Sia α2-3, rather than Sia α2-6 receptors, these findings support the contention that H1N1pdm viruses have a broader Sia receptor binding profile.⁸ Taken together, these observations suggest that H1N1pdm virus differs in subtle but important ways from seasonal influenza viruses in receptor usage and tissue tropism, and this may be important in its pathogenesis and transmission.Cytokine dysregulation is believed to contribute to the pathogenesis of human disease caused by HPAI H5N1 as well as the 1918 pandemic H1N1 viruses.^{10,11,12,13,14} It is not known whether the H1N1pdm virus differs from seasonal influenza in the induction of proinflammatory host responses in human tissues. The lungs of H1N1pdm-infected mice had a markedly different cytokine profile when compared with seasonal influenza infected animals with elevated levels of interleukin (IL)-4, IL-10, and interferon (IFN)-γ. The lungs of H1N1pdm-infected macaques also had higher levels of chemokines MCP-1, MIP-1α, IL-6, and IL-18.⁶ However, it is not known whether these host responses simply reflect the greater or more extensive replication of the H1N1pdm virus in the lung when compared with seasonal influenza viruses or are attributable to intrinsic differences in the virus itself being able to induce a more potent innate host response as occurs with the highly pathogenic avian influenza H5N1 virus. When primary human cells (macrophages and type I-like pneumocytes) are infected with seasonal and HPAI H5N1 influenza viruses of comparable infectious titers, the HPAI H5N1 viruses differentially hyperinduce a range of proinflammatory responses over a single virus replication cycle.^10,11,14 Thus it is clear that the H5N1 virus has inherent properties that lead to an exaggerated innate immune response. It is relevant to use a similar approach to investigate the host innate immune responses induced by pandemic H1N1pdm compared with that of seasonal influenza H1N1 virus in primary human respiratory epithelium.We have previously used ex vivo cultures of nasopharynx, tonsillar tissue, and lung for investigating virus tropism.¹⁵ We have also established in vitro cultures of polarized primary human respiratory epithelial cells, including type I–like alveolar epithelial cells, nasopharyngeal epithelial cells, and differentiated bronchial epithelial cells for investigating tissue tropism and innate immune host responses elicited by influenza viruses.^10,14,15 These in vitro cultures of bronchial epithelium differentiated at an air–liquid interface (ALI) provide a good representation of the human bronchial epithelium and have a ciliated epithelium as well as mucus producing goblet cells. We have also recently established ex vivo tissue culture models of human conjunctival epithelium. We now use these ex vivo human tissue cultures as well as the primary human respiratory epithelial cell cultures to compare the virus replication competence, cell tropism, and host innate immune responses of the pandemic H1N1pdm virus with that of seasonal influenza H1N1 viruses and, where relevant, avian HPAI H5N1 and H7N7 viruses.We demonstrate that whereas seasonal H1N1 and pandemic H1N1pdm viruses replicate comparably in ex vivo cultures of human nasopharynx and lung tissues, the human conjunctiva is preferentially infected by H1N1pdm rather than seasonal influenza H1N1 or H3N2 viruses. Pandemic H1N1pdm replicates more efficiently than seasonal H1N1 virus in differentiated bronchial epithelial cells in vitro at 33°C, but the two viruses replicate comparably at 37°C. We also demonstrate that the pandemic H1N1pdm virus does not differ from the human seasonal influenza viruses in their ability to induce proinflammatory cytokines and therefore does not appear to have the same potential to induce cytokine dysregulation as that manifested by HPAI H5N1 or the 1918 H1N1 virus.     

The molecular epidemiology of varicella-zoster virus: evidence for geographic segregation

Of 75 varicella-zoster virus (VZV) isolates obtained from patients in Africa, Asia, and the Far East, 74 (98.6%) were found to be positive for a BglI restriction site in gene 54. By contrast, <22% of strains from patients in the United Kingdom and in North and South America were positive for the BglI restriction site. Viruses positive for BglI were significantly more common in zoster occurring in patients of nonwhite origin (P<.05). Irrespective of the country in which the sample was obtained, 98% of strains positive for BglI clustered within a single phylogenetic group, which we termed "group A"; the exception was 1 strain that appeared to be recombinant genotype C/A. We used the BglI site to examine both the spread of type A viruses in the United Kingdom and the patterns of VZV infections within persons from different ethnic groups who grew up in the United Kingdom or abroad.     

Brain-derived neurotrophic factor mRNA and protein in the piglet brainstem and effects of Intermittent Hypercapnic Hypoxia

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for the development of normal respiratory rhythm and ventilatory control. Chronic exposure to Intermittent Hypercapnic Hypoxia (IHH) has been shown to alter ventilatory responses of piglets. This study investigated changes in BDNF distribution and expression in seven nuclei of the caudal medulla, from piglets exposed to IHH for 1, 2, 3, or 4 days before death, using non-radioactive in situ hybridisation (for mRNA) and immunohistochemistry (for protein). Compared to controls, BDNF mRNA was markedly increased across the entire medulla of the brainstem, after all durations of IHH (1-4 days). In contrast, BDNF protein expression increased after 1 day of exposure to IHH (p=0.003), but, thereafter, was not different to controls. Amongst individual nuclei, neurons of the dorsal motor nucleus of the vagus (DMNV) showed increased BDNF mRNA (p<0.01), but decreased protein expression (p=0.05) after all durations of IHH. In the ION, both mRNA and protein for BDNF were significantly increased after 1 day IHH (p<0.01 and p=0.001, respectively), but these increases were not sustained. This study is the first to investigate changes in BDNF expression in response to environmental challenges during postnatal development in the brainstem. Implications of the wide distribution of BDNF in the piglet caudal medulla and increased expression after IHH exposure are discussed, with particular reference to roles for BDNF-dependent neurons at this stage of development.     

Viral loads in clinical specimens and SARS manifestations

A retrospective viral load study was performed on clinical specimens from 154 patients with laboratory-confirmed severe acute respiratory syndrome (SARS); the specimens were prospectively collected during patients' illness. Viral load in nasopharyngeal aspirates (n = 142) from day 10 to day 15 after onset of symptoms was associated with oxygen desaturation, mechanical ventilation, diarrhea, hepatic dysfunction, and death. Serum viral load (n = 53) was associated with oxygen desaturation, mechanical ventilation, and death. Stool viral load (n = 94) was associated with diarrhea, and urine viral load (n = 111) was associated with abnormal urinalysis results. Viral replications at different sites are important in the pathogenesis of clinical and laboratory abnormalities of SARS.     

Prolonged disturbances of in vitro cytokine production in patients with severe acute respiratory syndrome (SARS) treated with ribavirin and steroids

Severe acute respiratory syndrome (SARS) is a new disease which has spread rapidly and widely. We wished to know whether evaluation of in vitro cytokine production could contribute to improved understanding of disease pathogenesis and to better patient management. Numbers of unstimulated and mitogen-stimulated cytokine-secreting peripheral blood mononuclear cells were measured repeatedly during and after hospitalization in 13 patients with SARS using enzyme-linked immunospot technology. Numbers of interferon-gamma, interleukin (IL)-2, IL-4, IL-10 and IL-12 secreting cells induced by T cell activators were below normal in many or most patients before and during treatment with corticosteroids and ribavirin but returned essentially to normal after completion of treatment. Staphylococcus aureus Cowan 1 (SAC)-stimulated IL-10 secreting cells were increased in early SARS but fell during treatment. SAC-induced IL-12 secreting cells were deficient before, during and long after treatment. Numbers of cells induced to produce IL-6 and tumour necrosis factor-alpha by T cell or monocyte activators were higher than normal in many early SARS patients and were still increased in some during and after treatment. We conclude that prolonged dysregulated cytokine production occurs in SARS and that future studies should be directed at improving anti-inflammatory and antiviral therapies in order to limit cytokine impairment.     

Cardiopathia fantastica: the cardiac variant of Munchausen syndrome

Munchausen syndrome is a rare condition in which the patient repeatedly seeks medical care for factitious illnesses. Cardiac Munchausen syndrome was first reported in 1953 and later referred to as cardiopathia fantastica. It is characterized by clinical manifestations of acute cardiac disease that are feigned and recurrent. Cardiopathia fantastica can have a variety of presentations similar to true cardiac disease. Most cases have presented with chest pain simulating acute coronary artery disease, but arrhythmias, hypertensive crises, abnormal biochemistry, and electrocardiographic findings have also been noted. These patients are willing to undergo expensive, invasive, and risky procedures to evaluate their simulated illness. This condition is likely to be significantly underreported. In some patients, the presence of abnormal findings that are clinically insignificant may complicate the investigative approach. Patients with this disorder consume a disproportionate amount of health care dollars and sometimes are left with residual deficits as complications of invasive procedures. In this review, we discuss the recognition, manifestations, and treatment of cardiopathia fantastica.     

Insulin: a novel factor in carcinogenesis

Cancer is a leading cause of mortality in the United States. Despite much research on specific carcinogens, the cause of many cancers remains unclear. The identification of novel causative agents offers the potential for cancer prevention. Diseases such as obesity and diabetes mellitus, characterized by hyperinsulinemia, are associated with increased risk of endometrial, colorectal, and breast carcinomas. There is increasing evidence that insulin is a growth factor for tumor formation. The mechanisms underlying insulin-mediated neoplasia may include enhanced DNA synthesis with resultant tumor cell growth, inhibition of apoptosis, and altered sex hormone milieu. The reduced insulin levels seen with physical activity, weight loss, and a high fiber diet may account for decreased cancer risk. The role of newer drugs that restore sensitivity to insulin, thereby reducing hyperinsulinemia, is an exciting potential area of cancer prevention. In this review, we discuss the potential role of insulin as a tumor growth factor.