Receptor binding properties, cell tropism and transmission of influenza A virus

The first influenza pandemic of the 21st century was caused by the influenza A (H1N1) 2009 virus (A(H1N1)pdm09) that emerged from a swine-origin source. Although human infections with swine-origin influenza have been reported intermittently in the past decade, none went on to cause a pandemic or sustained human-to-human transmission. In previous pandemics, specific residues in the receptor binding site of the haemagglutinin (HA) protein of influenza have been associated with the ability of the virus to transmit between humans. In this thesis the effect of mutations at residue 227 in HA on cell tropism and transmission of A(H1N1)pdm09 is described. In A(H1N1)pdm09 and previous seasonal H1N1 viruses this residue is glutamic acid (E), whereas in swine influenza it is alanine (A). Using human airway epithelium, a differential cell tropism of A(H1N1)pdm09 compared to A(H1N1)pdm09 E227A and swine influenza was shown suggesting this residue may alter the sialic acid conformer binding preference of the HA. Furthermore, multi-cycle viral growth of both A(H1N1)pdm09 E227A and swine influenza was found to be attenuated in comparison to A(H1N1)pdm09 in human airway epithelium. However this altered tropism and viral growth in human airway epithelium did not abrogate respiratory droplet transmission of A(H1N1)pdm09 E227A in ferrets. This suggests that acquisition of 227E was not solely responsible for the ability of A(H1N1)pdm09 to transmit between humans. Because the work with the E227A mutant showed that small differences in cell tropism that may affect influenza virus transmissibility could be detected in human airway cells, a receptor binding assay was developed for laboratory surveillance using commercial human airway epithelium cultures, to screen for zoonotic influenza strains of particular concern for human health. To further investigate adaptations by influenza virus for infection of the human host, the cell tropism determined by the HA protein of an avian H7N7 and human H7N3 isolate was compared. Binding to non-ciliated human airway cells was increased for the human isolate. This human case of H7N3 infection yielded two isolates from different sites (eye and throat) from the same patient on the same day. A whole genome sequencing assay was designed for H7 isolates and both eye and throat isolate were fully sequenced. One synonymous nucleotide change was found in the NS gene segment and one synonymous and two non-synonymous nucleotide changes were found in the PB2 gene segment. Comparison of the non-synonymous changes in the protein sequence of PB2 to available avian and human virus PB2 sequences revealed that the substitutions in the eye isolate were comparatively uncommon. Interestingly, these changes resulted in an increased viral growth in human airway epithelial cells at 32ºC when compared to the throat isolate, a phenomenon which was not observed at 37ºC. Finally, the use of a lung model maintained by the ex-vivo lung perfusion (EVLP) technique for study of virus infection was tested. This technique allows the use of both human and porcine lungs up to 24 hours after abstraction and is a potential model for respiratory pathogens and novel treatments. Porcine lungs were infected with A(H1N1)pdm09. Physiological and virological parameters were measured in two separate experiments and infection was demonstrated by increased viral loads in samples obtained at late time points after infection

Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012. Recently, the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific interaction of the receptor-binding domain (RBD) of MERS-CoV spike protein and DPP4 was determined by crystallography. Animal studies identified rhesus macaques but not hamsters, ferrets, or mice to be susceptible for MERS-CoV. Here, we investigated the role of DPP4 in this observed species tropism. Cell lines of human and nonhuman primate origin were permissive of MERS-CoV, whereas hamster, ferret, or mouse cell lines were not, despite the presence of DPP4. Expression of human DPP4 in nonsusceptible BHK and ferret cells enabled MERS-CoV replication, whereas expression of hamster or ferret DPP4 did not. Modeling the binding energies of MERS-CoV spike protein RBD to DPP4 of human (susceptible) or hamster (nonsusceptible) identified five amino acid residues involved in the DPP4-RBD interaction. Expression of hamster DPP4 containing the five human DPP4 amino acids rendered BHK cells susceptible to MERS-CoV, whereas expression of human DPP4 containing the five hamster DPP4 amino acids did not. Using the same approach, the potential of MERS-CoV to utilize the DPP4s of common Middle Eastern livestock was investigated. Modeling of the DPP4 and MERS-CoV RBD interaction predicted the ability of MERS-CoV to bind the DPP4s of camel, goat, cow, and sheep. Expression of the DPP4s of these species on BHK cells supported MERS-CoV replication. This suggests, together with the abundant DPP4 presence in the respiratory tract, that these species might be able to function as a MERS-CoV intermediate reservoir

Infection with Mers-Cov Causes Lethal Pneumonia in the Common Marmoset

The availability of a robust disease model is essential for the development of countermeasures for Middle East respiratory syndrome coronavirus (MERS-CoV). While a rhesus macaque model of MERS-CoV has been established, the lack of uniform, severe disease in this model complicates the analysis of countermeasure studies. Modeling of the interaction between the MERS-CoV spike glycoprotein and its receptor dipeptidyl peptidase 4 predicted comparable interaction energies in common marmosets and humans. The suitability of the marmoset as a MERS-CoV model was tested by inoculation via combined intratracheal, intranasal, oral and ocular routes. Most of the marmosets developed a progressive severe pneumonia leading to euthanasia of some animals. Extensive lesions were evident in the lungs of all animals necropsied at different time points post inoculation. Some animals were also viremic; high viral loads were detected in the lungs of all infected animals, and total RNAseq demonstrated the induction of immune and inflammatory pathways. This is the first description of a severe, partially lethal, disease model of MERS-CoV, and as such will have a major impact on the ability to assess the efficacy of vaccines and treatment strategies as well as allowing more detailed pathogenesis studies

SARS-Like coronavirus WIV1-CoV does not replicate in Egyptian fruit bats (Rousettus aegyptiacus)

Severe acute respiratory syndrome (SARS)-like WIV1-coronavirus (CoV) was first isolated from Rhinolophus sinicus bats and can use the human angiotensin converting enzyme 2 (ACE2) receptor. In the current study, we investigate the ability of WIV1-CoV to infect Rousettus aegyptiacus bats. No clinical signs were observed throughout the experiment. Furthermore, only four oropharyngeal swabs and two respiratory tissues, isolated on day 3 post inoculation, were found positive for viral RNA. Two out of twelve bats showed a modest increase in coronavirus specific antibodies post challenge. In conclusion, WIV1-CoV was unable to cause a robust infection in Rousettus aegyptiacus bats

Risk Factors for Middle East Respiratory Syndrome Coronavirus Infection among Camel Populations, Southern Jordan, 2014-2018.

After the first detection of Middle East respiratory syndrome coronavirus (MERS-CoV) in camels in Jordan in 2013, we conducted 2 consecutive surveys in 2014-2015 and 2017-2018 investigating risk factors for MERS-CoV infection among camel populations in southern Jordan. Multivariate analysis to control for confounding demonstrated that borrowing of camels, particularly males, for breeding purposes was associated with increased MERS-CoV seroprevalence among receiving herds, suggesting a potential route of viral transmission between herds. Increasing age, herd size, and use of water troughs within herds were also associated with increased seroprevalence. Closed herd management practices were found to be protective. Future vaccination strategies among camel populations in Jordan could potentially prioritize breeding males, which are likely to be shared between herds. In addition, targeted management interventions with the potential to reduce transmission between herds should be considered; voluntary closed herd schemes offer a possible route to achieving disease-free herds

Mechanistic theory predicts the effects of temperature and humidity on inactivation of SARS-CoV-2 and other enveloped viruses

Ambient temperature and humidity strongly affect inactivation rates of enveloped viruses, but a mechanistic, quantitative theory of these effects has been elusive. We measure the stability of SARS-CoV-2 on an inert surface at nine temperature and humidity conditions and develop a mechanistic model to explain and predict how temperature and humidity alter virus inactivation. We find SARS-CoV-2 survives longest at low temperatures and extreme relative humidities (RH); median estimated virus half-life is >24 hr at 10°C and 40% RH, but ∼1.5 hr at 27°C and 65% RH. Our mechanistic model uses fundamental chemistry to explain why inactivation rate increases with increased temperature and shows a U-shaped dependence on RH. The model accurately predicts existing measurements of five different human coronaviruses, suggesting that shared mechanisms may affect stability for many viruses. The results indicate scenarios of high transmission risk, point to mitigation strategies, and advance the mechanistic study of virus transmission

Effectiveness of N95 respirator decontamination and reuse against SARS-CoV-2 virus

The coronavirus pandemic has created worldwide shortages of N95 respirators. We analyzed 4 decontamination methods for effectiveness in deactivating severe acute respiratory syndrome coronavirus 2 virus and effect on respirator function. Our results indicate that N95 respirators can be decontaminated and reused, but the integrity of respirator fit and seal must be maintained

Evaluation of Five Buffers for Inactivation of Monkeypox Virus and Feasibility of Virus Detection Using the Panther Fusion® Open Access System

Rapid diagnosis is key to containing viral outbreaks. However, for the current monkeypox outbreak the major deterrent to rapid testing is the requirement for higher biocontainment of potentially infectious monkeypox virus specimens. The current CDC guidelines require the DNA extraction process before PCR amplification to be performed under biosafety level 3 unless vaccinated personnel are performing assays. This increases the turn-around time and makes certain laboratories insufficiently equipped to handle specimens from patients with suspected monkeypox infection. We investigated the ability of five commercially available lysis buffers and heat for inactivation of monkeypox virus. We also optimized the use of monkeypox virus in Hologic® Panther Specimen Lysis Buffer for detection of virus in the Panther Fusion® Open Access System using published generic and clade specific monkeypox virus primers and probes