Anna J. X. Zhang

Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats

We describe the discovery and isolation of a paramyxovirus, feline morbillivirus (FmoPV), from domestic cat (Felis catus). FmoPV RNA was detected in 56 (12.3%) of 457 stray cats (53 urine, four rectal swabs, and one blood sample) by RT-PCR. Complete genome sequencing of three FmoPV strains showed genome sizes of 16,050 bases, the largest among morbilliviruses, because of unusually long 5′ trailer sequences of 400 nt. FmoPV possesses identical gene contents (3′-N-P/V/C-M-F-H-L-5′) and is phylogenetically clustered with other morbilliviruses. IgG against FmoPV N protein was positive in 49 sera (76.7%) of 56 RT-PCR–positive cats, but 78 (19.4%) of 401 RT-PCR–negative cats (P < 0.0001) by Western blot. FmoPV was isolated from CRFK feline kidney cells, causing cytopathic effects with cell rounding, detachment, lysis, and syncytia formation. FmoPV could also replicate in subsequent passages in primate Vero E6 cells. Infected cell lines exhibited finely granular and diffuse cytoplasmic fluorescence on immunostaining for FmoPV N protein. Electron microscopy showed enveloped virus with typical “herringbone” appearance of helical N in paramyxoviruses. Histological examination of necropsy tissues in two FmoPV-positive cats revealed interstitial inflammatory infiltrate and tubular degeneration/necrosis in kidneys, with decreased cauxin expression in degenerated tubular epithelial cells, compatible with tubulointerstitial nephritis (TIN). Immunohistochemical staining revealed FmoPV N protein-positive renal tubular cells and mononuclear cells in lymph nodes. A case-control study showed the presence of TIN in seven of 12 cats with FmoPV infection, but only two of 15 cats without FmoPV infection (P < 0.05), suggesting an association between FmoPV and TIN.

D225G mutation in hemagglutinin of pandemic influenza H1N1 (2009) virus enhances virulence in mice.

Although the majority of infections by the pandemic influenza H1N1 (2009) virus is mild, a higher mortality occurs in young adults with no risk factors for complications. Some of these severe cases were infected by the virus with an aspartate to glycine substitution at 225 position (D225G, H3 numbering) in the hemagglutinin (HA). Previous studies with the highly virulent 1918 pandemic H1N1 virus suggested that such substitution was associated with a dual binding specificity of the virus for both α2,3- and α2,6-linked sialic acid receptors on host cells. Thus, the D225G mutant may cause more severe disease with its increased predilection for the lower respiratory tract, where the α2,3 sialic acid receptor is more prevalent, but this hypothesis has not been investigated. We obtained a mutant virus after four sequential passages in lungs of BALB/c mice with a wild-type pandemic influenza A H1N1 (2009) virus. One plaque purified mutant virus had a single non-synonymous D225G mutation in the HA gene. This mutant was more lethal to chick embryo and produced a viral load of about two log higher than that of the wild-type parental virus during the first 24 h. A pathogenicity test showed that the 50% lethal dose in mice (LD50) was reduced from over 2 × 106 plaque-forming units (PFU) with the parental virus to just 150 PFU with the mutant virus. The survival of mice challenged with the mutant virus was significantly decreased when compared with the parental virus (P < 0.0001). Significantly higher viral titers and elevated proinflammatory cytokines in lung homogenates of mice infected with the mutant virus were found, which were compatible with severe histopathological changes of pneumonitis. The only consistent mutation in the genomes of viral clones obtained from dying mice was D225G substitution.

Wild type and mutant 2009 pandemic influenza A (H1N1) viruses cause more severe disease and higher mortality in pregnant BALB/c mice.

Background Pregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear. Principal Findings We showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes. Conclusion The adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation.

High Titer and Avidity of Nonneutralizing Antibodies against Influenza Vaccine Antigen Are Associated with Severe Influenza

ABSTRACT The importance of neutralizing antibody in protection against influenza virus is well established, but the role of the early antibody response during the initial stage of infection in affecting the severity of disease is unknown. The 2009 influenza pandemic provided a unique opportunity for study because most patients lacked preexisting neutralizing antibody. In this study, we compared the antibody responses of 52 patients with severe or mild disease, using sera collected at admission. A microneutralization (MN) assay was used to detect neutralizing antibody. We also developed an enzyme-linked immunosorbent assay (ELISA) which detects both neutralizing and nonneutralizing antibodies against viral antigens from a split-virion inactivated monovalent influenza virus vaccine. While the MN titers were not significantly different between the two groups (P = 0.764), the ELISA titer and ELISA/MN titer ratio were significantly higher for patients with severe disease than for those with mild disease (P = 0.004 and P = 0.011, respectively). This finding suggested that in patients with severe disease, a larger proportion of serum antibodies were antibodies with no detectable neutralizing activity. The antibody avidity was also significantly higher in patients with severe disease than in those with mild disease (P < 0.05). Among patients with severe disease, those who required positive pressure ventilation (PPV) had significantly higher ELISA titers than those who did not require PPV (P < 0.05). Multivariate analysis showed that the ELISA titer and antibody avidity were independently associated with severe disease. Higher titers of nonneutralizing antibody with higher avidity at the early stage of influenza virus infection may be associated with worse clinical severity and poorer outcomes.

Leptin Mediates the Pathogenesis of Severe 2009 Pandemic Influenza A(H1N1) Infection Associated With Cytokine Dysregulation in Mice With Diet-Induced Obesity

BACKGROUND Obesity is associated with a high circulating leptin level and severe 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) infection. The mechanism for severe lung injury in obese patients and the specific treatment strategy remain elusive. METHOD We studied the pathogenesis of A(H1N1)pdm09 infection in a mouse model of diet-induced obesity. RESULTS Obese mice had significantly higher initial pulmonary viral titer and mortality after challenge with A(H1N1)pdm09, compared with age-matched lean mice. Compared with lean mice, obese mice had heightened proinflammatory cytokine and chemokine levels and more severe pulmonary inflammatory damage. Furthermore, obese mice had a higher preexisting serum leptin level but a lower preexisting adiponectin level. Recombinant mouse leptin increased the interleukin 6 (IL-6) messenger RNA expression in mouse single-lung-cell preparations, mouse macrophages, and mouse lung epithelial cell lines infected with A(H1N1)pdm09. Administration of anti-leptin antibody improved the survival of infected obese mice, with associated reductions in pulmonary levels of the proinflammatory cytokines IL-6 and interleukin 1β but not the pulmonary viral titer. CONCLUSIONS Our findings suggest that preexisting high levels of circulating leptin contribute to the development of severe lung injury by A(H1N1)pdm09 in mice with diet-induced obesity. The therapeutic strategy of leptin neutralization for the reduction of proinflammatory responses and pulmonary damage in obese patients warrants further investigations.

Differences in Antibody Responses of Individuals with Natural Infection and Those Vaccinated against Pandemic H1N1 2009 Influenza

ABSTRACT The differential antibody response measured by the commonly used hemagglutination inhibition (HI) and microneutralization (MN) assays in patients with natural infection and vaccination has not been fully assessed. HI and conventional MN (CMN) assays were performed on sera from 651 patients with natural infection by pandemic H1N1 2009 influenza virus and on sera from 567 recipients of the corresponding vaccine. Surprisingly, the overall seroprotection rates determined by CMN and HI assays in vaccine recipients were only 44.8 and 35.1%, respectively. Antibody titers measured by the CMN assay was significantly higher than that obtained by HI assay in vaccine recipients aged ≥50 years, but these titers were not significantly different among younger vaccine recipients. In contrast, the HI titer was greater than the CMN titer for the age group from 16 to 29 years but was not significantly different in other age groups for natural infection. Lower antibody levels were found in both naturally infected patients and immunized recipients in the older than in the younger age groups, but naturally infected patients exhibited higher HI and CMN titers than did the corresponding vaccine recipients. In addition, we developed a rapid fluorescent focus microneutralization (FFMN) assay to test sera from naturally infected patients. The FFMN assay has a better correlation with CMN than with HI (ρ = 0.810 versus 0.684), which is expected of neutralizing antibody mainly targeted toward the inhibition of viral entry into cells. The higher antibody level elicited by natural infection than by vaccination may be related to differences between antigen presentation by the intramuscular route of vaccination and mucosal viral replication in mucosal cells of the respiratory tract.

A Novel Psittacine Adenovirus Identified During an Outbreak of Avian Chlamydiosis and Human Psittacosis: Zoonosis Associated with Virus-Bacterium Coinfection in Birds

Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3′ end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3–54.0% for the DNA polymerase, 64.6–70.7% for the penton protein, and 66.1–74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be emphasized in the investigation of disease outbreaks in human and animals.

Toll-Like Receptor 7 Agonist Imiquimod in Combination with Influenza Vaccine Expedites and Augments Humoral Immune Responses against Influenza A(H1N1)pdm09 Virus Infection in BALB/c Mice

ABSTRACT Toll-like receptors (TLRs) of the innate immune system are known targets for enhancing vaccine efficacy. We investigated whether imiquimod, a synthetic TLR7 agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccine. BALB/c mice were immunized intraperitoneally with monovalent A(H1N1)pdm09 vaccine combined with imiquimod (VCI) prior to intranasal inoculation with a lethal dose of mouse-adapted A(H1N1)pdm09 virus. For mice immunized 3 days before infection, the survival rates were significantly higher in the VCI group (60%, mean survival time[MST], 11 days) than in the vaccine-alone (30%; MST, 8.8 days), imiquimod-alone (5%; MST, 8.4 days), and phosphate-buffered saline (PBS) (0%; MST, 6.2 days) groups (P < 0.01). In the VCI group, 45 and 35% of the mice survived even when they were infected 2 days or 1 day after immunization. Virus-specific serum IgM, IgG, and neutralizing antibodies appeared earlier with higher geometric mean titers in the VCI group than in the control groups. The pulmonary viral load was significantly lower at all time points postinfection in the VCI, vaccine-alone, and imiquimod-alone groups than in the PBS control group (P < 0.05). The protection induced by VCI was specific for A(H1N1)pdm09 virus but not for A(H5N1) virus. Since imiquimod combined with RNase-treated vaccine is as protective as imiquimod combined with untreated vaccine, mechanisms other than TLR7 may operate in expediting and augmenting immune protection. Moreover, increased gamma interferon mRNA expression and IgG isotype switching, which are markers of the Th1 response induced by imiquimod, were not apparent in our mouse model. The mechanisms of imiquimod-induced immune protection deserve further study.

High Incidence of Severe Influenza among Individuals over 50 Years of Age

ABSTRACT Age-specific epidemiological data on asymptomatic, symptomatic, and severe infections are essential for public health policies on combating influenza. In this study, we incorporated data on microbiologically confirmed infections and seroprevalence to comprehensively describe the epidemiology of pandemic H1N1 2009 influenza. Seroprevalence was determined from 1,795 random serum samples collected in our hospital in January 2007 (before the first wave of the pandemic) and March 2010 (after the second wave). Data on microbiologically confirmed infection and severe cases were obtained from the Centre for Health Protection in Hong Kong. Severe cases were most common in the 51- to 60-year-old age group. The microbiologically confirmed incidence rate was highest for children aged ≤10 years and dropped sharply for the adult population (ρ = −1.0; P < 0.01), but the incidence rate for severe disease was highest for the 51- to 60-year-old age group. For the 51- to 60-year-old age group, the seroprevalence was similar to that for the younger age groups, but the proportion of severe cases relative to seroprevalence was significantly higher than that for 11- to 50-year-old age groups. As judged from the percentage of specimens positive for other respiratory viruses compared with that for pandemic H1N1 virus, the impact of symptomatic disease due to pandemic H1N1 virus was higher than that for other respiratory viruses in people aged ≤50 years. In conclusion, the 51- to 60-year-old age group, which had the highest overall incidence and the highest rate of severe disease but is currently not considered by the World Health Organization to be an at-risk group, should be prioritized for influenza vaccination in areas where universal influenza vaccination is not practiced.

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

Background The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). Methodology/Principal Findings The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Conclusions/Significance Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.