Sparse evidence of MERS‐CoV infection among animal workers living in Southern Saudi Arabia during 2012

Middle East respiratory syndrome coronavirus (MERS‐CoV) is an emerging viral pathogen that primarily causes respiratory illness. We conducted a seroprevalence study of banked human serum samples collected in 2012 from Southern Saudi Arabia. Sera from 300 animal workers (17% with daily camel exposure) and 50 non‐animal‐exposed controls were examined for serological evidence of MERS‐CoV infection by a pseudoparticle MERS‐CoV spike protein neutralization assay. None of the sera reproducibly neutralized the MERS‐CoV‐pseudotyped lentiviral vector. These data suggest that serological evidence of zoonotic transmission of MERS‐CoV was not common among animal workers in Southern Saudi Arabia during July 2012.     

Adenovirus respiratory infection in hospitalized children in Hong Kong: serotype–clinical syndrome association and risk factors for lower respiratory tract infection

Lower respiratory tract infections (LRTI) caused by adenovirus can be severe with resultant chronic pulmonary sequelae. More than 50 serotypes have been recognized; however, the exact association of serotype with clinical phenotype is still unclear. There have been no reports on the adenovirus serotype pattern in Hong Kong, and their relationships with disease manifestations and complications are not known. Clinical and epidemiological data on 287 children (<6 years old) admitted with adenovirus respiratory infections from 2001 to 2004 were reviewed. Common presenting symptoms included fever (97.9 %) and cough and rhinitis (74 %). Extra-pulmonary manifestations were present in 37.3 %. The clinical picture mimicked bacterial infection for its prolonged high fever and neutrophilic blood picture. Forty-two patients (14.6 %) had LRTI, either pneumonia or acute bronchiolitis, but none had severe acute respiratory compromise. Children aged 1 to 2 years old were most at risk for adenovirus LRTI (adjusted p?=?0.0165). Serotypes 1 to 7 could be identified in 93.7 % of the nasopharyngeal specimens, with serotypes 2 and 3 being the most prevalent. Different serotypes showed predilection for different age groups and with different respiratory illness association. The majority of acute bronchiolitis (71.4 %) were associated with serotype 2 infection, and this association was statistically significant (p?<?0.0001). Serotype 3 infection accounted for over half of the pneumonia cases (57–75 %) in those aged 3–5 years old. Only one patient developed mild bronchiectasis after serotype 7 pneumonia. Children aged 1 to 2 years old were the at-risk group for adenovirus LRTI, but respiratory morbidity was relatively mild in our locality. There was an apparent serotype–respiratory illness association.     

IL-15 adjuvanted multivalent vaccinia-based universal influenza vaccine requires CD4+ T cells for heterosubtypic protection

Current influenza vaccines are ineffective against novel viruses and the source or the strain of the next outbreak of influenza is unpredictable; therefore, establishing universal immunity by vaccination to limit the impact of influenza remains a high priority. To meet this challenge, a novel vaccine has been developed using the immunogenic live vaccinia virus as a vaccine vector, expressing multiple H5N1 viral proteins (HA, NA, M1, M2, and NP) together with IL-15 as a molecular adjuvant. Previously, this vaccine demonstrated robust sterile cross-clade protection in mice against H5 influenza viruses, and herein its use has been extended to mediate heterosubtypic immunity toward viruses from both group 1 and 2 HA lineages. The vaccine protected mice against lethal challenge by increasing survival and significantly reducing lung viral loads against the most recent human H7N9, seasonal H3N2, pandemic-2009 H1N1, and highly pathogenic H7N7 influenza A viruses. Influenza-specific antibodies elicited by the vaccine failed to neutralize heterologous viruses and were unable to confer protection by passive transfer. Importantly, heterologous influenza-specific CD4⁺ and CD8⁺ T-cell responses that were elicited by the vaccine were effectively recalled and amplified following viral challenge in the lungs and periphery. Selective depletion of T-cell subsets in the immunized mice revealed an important role for CD4⁺ T cells in heterosubtypic protection, despite low sequence conservation among known MHC-II restricted epitopes across different influenza viruses. This study illustrates the potential utility of our multivalent Wyeth/IL-15/5Flu as a universal influenza vaccine with a correlate of protective immunity that is independent of neutralizing antibodies.Influenza causes widespread infection during seasonal epidemics and occasional worldwide pandemics despite available vaccines. The subtype of future outbreaks or pandemic influenza strains is unpredictable as is its source, evident from the most recent H7N9 outbreak from poultry in China, the variant H3N2 outbreak from swine in the United States in 2012, the H1N1 worldwide pandemic of 2009, and the highly pathogenic avian influenza (HPAI) H5N1 in 1997 from domestic poultry. Therefore, the development of a successful universal vaccination strategy is urgently needed.Universal protection requires heterosubtypic immunity (HSI), whereby vaccination against one influenza virus cross-protects against novel and emerging strains that could potentially be mediated by multiple adaptive immune mechanisms. T cells are potent mediators of HSI, because these cells typically recognize peptide epitopes derived from internal proteins of influenza virus, which are naturally more conserved than surface HA and NA across different strains and even serologically distinct viral subtypes. Because of sequence conservation of the majority of T-cell epitopes between different influenza viruses, cross-reactive T-cell responses have been detected in healthy seronegative individuals against H5N1 and pandemic H1N1-2009 viruses (1, 2).Previously, we generated a multivalent vaccinia virus-based H5N1 influenza vaccine, which demonstrated effective cross-clade immunity against lethal H5N1 challenges. This vaccine expresses five H5N1-derived influenza proteins (HA, NA, M1, M2, and NP), in combination with the immune stimulatory cytokine IL-15 (3) that increases long-term memory responses, along with enhanced T-cell, B-cell, and NK cell functions, including cytokine production and survival. Despite the use of live vaccine vectors being generally constrained in immune-compromised individuals, the vaccine vector (Wyeth/IL-15) has been proven safe and effective in primates and immune-deficient mice (4).A cell-culture–derived, live vaccine vector that encodes full-length influenza proteins with inherent capacity to access MHC-I and II processing pathways to establish robust influenza-specific T-cell responses is an excellent approach to overcome issues related to population-wide MHC polymorphism and egg-based production methods for HPAI vaccines. Herein, we extend the use of Wyeth/IL-15/5Flu vaccine against multiple human influenza viruses of different HA subtypes, including the highly pathogenic H7N7, pandemic H1N1-2009, seasonal H3N2, and the most recent human H7N9 viruses. The vaccine proved effective against all of the heterologous strains tested, and the immunological mechanisms of protection were investigated to decipher correlates of immunity.     

Validation of self-swab for virologic confirmation of influenza virus infections in a community setting

Few studies have investigated the validity of self-collected nose and throat swabs for influenza confirmation in community settings. We followed outpatients with confirmed influenza with sequential measurement of viral loads and applied log-linear regression models to the viral shedding patterns. Among 176 outpatients with confirmed influenza, the detection of virus and quantitative viral loads obtained from self-swabs was consistent with statistical predictions based on earlier and later measurements, suggesting that self-collected nose and throat swabs can be a valid alternative for virologic confirmation of influenza A or B infection in a community setting.     

Infection of swine ex vivo tissues with avian viruses including H7N9 and correlation with glycomic analysis

Objectives

Swine have been regarded as intermediate hosts in the spread of influenza from birds to humans but studies of the sialylated glycans that comprise their respiratory tract have not been extensively studied in the past. This study analyzed the sialylated N‐glycan and O‐glycan profile of swine trachea and lung and correlated this with ex‐vivo infection of swine explants with avian influenza viruses.

Sample

Lungs and tracheal samples were obtained from normal farm and laboratory raised swine and used for ex vivo infection as well as mass spectrometric analysis. Infection of the ex vivo tissues used high pathogenic and low pathogenic avian viruses including the novel H7N9 virus that emerged in China in early 2013.

Main outcome measures

Assessment of successful replication was determined by TCID50 as well as virus immunohistochemistry. The N‐glycan and O‐glycan profiles were measured by MALDI‐TOF and sialylated linkages were determined by sialidase treatment. Lectin binding histochemistry was also performed on formalin fixed tissue samples with positive binding detected by chromogen staining.

Results

The swine respiratory tract glycans differed from the human respiratory tact glycans in two main areas. There was a greater abundance of Gal‐α‐Gal linkages resulting in a relative decrease in sialylated glycans. The swine respiratory tract also had a greater proportion of glycans containing Neu5Gc and Siaα2‐6 glycans than the human respiratory tract. Infection with avian viruses was confined primarily to lung bronchioles rather than trachea and parenchyma.

Conclusions

In contrast to previous studies we found that there was not as much expression of Siaα2‐3 glycans on the surface of the trachea. Infection of Siaα2‐3 binding avian viruses was restricted to the lower respiratory tract bronchioles. This finding may diminish the ability of the swine to act as an intermediary in the transmission of avian viruses to humans.     

SARS-CoV-2 in Quarantined Domestic Cats from COVID-19 Households or Close Contacts,Hong Kong,China

We tested 50 cats from coronavirus disease households or close contacts in Hong Kong, China, for severe acute respiratory syndrome coronavirus 2 RNA in respiratory and fecal samples. We found 6 cases of apparent human-to-feline transmission involving healthy cats. Virus genomes sequenced from 1 cat and its owner were identical.     

Hyperinduction of cyclooxygenase-2-mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza H5N1 infection

The mechanism for the pathogenesis of H5N1 infection in humans remains unclear. This study reveals that cyclooxygenase-2 (COX-2) was strongly induced in H5N1-infected macrophages in vitro and in epithelial cells of lung tissue samples obtained during autopsy of patients who died of H5N1 disease. Novel findings demonstrated that COX-2, along with tumor necrosis factor alpha and other proinflammatory cytokines were hyperinduced in epithelial cells by secretory factors from H5N1-infected macrophages in vitro. This amplification of the proinflammatory response is rapid, and the effects elicited by the H5N1-triggered proinflammatory cascade are broader than those arising from direct viral infection. Furthermore, selective COX-2 inhibitors suppress the hyperinduction of cytokines in the proinflammatory cascade, indicating a regulatory role for COX-2 in the H5N1-hyperinduced host proinflammatory cascade. These data provide a basis for the possible development of novel therapeutic interventions for the treatment of H5N1 disease, as adjuncts to antiviral drugs.     

Reliable universal RT-PCR assays for studying influenza polymerase subunit gene sequences from all 16 haemagglutinin subtypes

Realizable one-step RT-PCR assays specific for influenza PB2, PB1 and PA segments are described in this report. The designs of the consensus primers were based on more than five thousands polymerase genes derived from avian or mammalian viral strains. All the viral RNA tested in this study could be consistently amplified by the assays. The reaction products were specific and could be used for direct DNA sequencing. These assays might be useful tools to study the sequences of these genes.