Coronaviruses (CoVs) and influenza A viruses (IAVs) are well known for their ability to cross host species barriers and colonize a wide range of avian and mammalian host species, including humans. Their potential emergence upon zoonotic introduction poses a substantial threat to public health. Just over a century ago, the 1918 influenza pandemic caused by an avian origin H1N1 IAV affected one-third of the world’s population and led to over 50 million deaths. A few decades later in years 1957, 1968 and 2009, three other major IAV pandemics occurred, each of which resulted in severe consequences for global economy and public health. In the meantime, apart from the established human coronaviruses, CoV outbreaks in humans occurred three times in the 21st century alone: the severe acute respiratory syndrome (SARS) CoV outbreak in 2002/2003, the Middle East respiratory syndrome CoV emergence in 2012 and the currently ongoing SARS-CoV-2 pandemic. Companion animals are also susceptible to CoVs and IAVs. In addition to animal health implications, the close contact between companion animals and humans also has potential risks of zoonotic virus infection, and reverse zoonosis of human CoVs and IAVs has been observed in these animals. Such facts emphasize the role of companion animals as potential virus mixing vessels, with the possibility of the emergence of recombinant viruses with increased threats for both animal and humans. Consequently, it is of importance to study CoV and IAV infection in companion animals, and recurrent surveillance studies are necessary.
Being classically defined as the studies of proteins found in bodily fluids, especially blood serum or plasma, serology was commonly used to study the prevalence of pathogen-specific antibodies in defined populations. Compared with molecular diagnostic methods at the acute stage of infection, serological methods have the advantage of being able to trace the infection after a longer period, as antibodies induced by viruses have long duration of persistence in blood after infection. Therefore, serological assays are crucial tools which can be applied to support clinical diagnosis of viral infection, monitoring of vaccine compliance, assessments of herd immunity and seroprevalence studies in different populations. In this thesis, we focused on the development of robust and specific serological assays that can serve as a toolkit for rapid diagnosis of CoV and IAV infections. This will give more insight and knowledge on the occurrence of different CoVs and IAVs infections in companion animals and their association with clinical diseases.
In cats, infections with feline coronaviruses (FCoVs) are common. Several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. However, whether cats might become naturally infected with CoVs of other species is unknown. In Chapter 2, we presented a thorough serological survey in cats using the S1 receptor binding subunit of the CoV spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. Recombinant CoV spike S1 proteins of different animal and human CoVs were used as antigens for screening of cat sera for the presence of antibodies against the respective proteins. Positive samples were further tested by virus neutralization assays. We observed antigenic cross-reactivity between S1s of type 1 and type 2 FCoVs, and between FCoV type 1 and porcine epidemic diarrhea virus. Domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the CD domains of S1. In addition, our observation that some feline sera displayed antibody reactivity exclusively against non-feline CoV S1 proteins warrant further research into the epidemiology and cross-species transmission of coronaviruses in cats and other animals that are in close contact with humans.
Epidemiological studies of equine coronavirus (ECoV) infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. Chapter 3 describes the development and validation of an S1-protein-based ELISA method for the detection of specific antibodies against ECoV. With this method, we are able to provide a consolidated diagnostic test to confirm ECoV outbreaks, as a complement to qRT-PCR analysis of equine feces samples. Our in-house ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.
In Chapter 4, we performed a survey to follow and study the spread of SARS-CoV-2 in companion animals. A set of serological assays including ELISA and virus neutralization were developed and validated and next used to conduct the first seroprevalence study in the Netherlands. The general prevalence of antibodies to SARS-CoV-2 in the animal population with unknown SARS-CoV-2 exposure included in our study is low but supports the importance of continuous serosurveillance of SARS-CoV-2 in these companion animals and a wider range of other animal species. This is especially important now the incidence of COVID-19 in humans is decreasing in several parts of the world.
Except for studies of CoVs, in Chapter 5, we developed a pipeline of serological assays which allow broad to specific analysis of IAV-specific antibody responses in cats and dogs. In this pipeline, serum samples were tested first with HA- and HA1-specific ELISAs and subsequently analyzed by nanoparticle-based, virus-free HI assays. Using these three assays, we found cat and dog sera from different cohorts to be positive for antibodies against one or more IAV subtypes and/or strains. Cat and dog serum samples collected after the 2009 pandemic H1N1 outbreak exhibit much higher seropositivity against H1 compared to samples from before 2009. Cat sera, furthermore, displayed higher reactivity for avian IAVs than dog sera. Our findings show the added value of using complementary serological assays, which are based on reactivity with different numbers of HA epitopes, to study IAV antibody responses and for improved serosurveillance of IAV infections. These observations highlight the role of cats and dogs in IAV ecology and indicate the potential of these companion animals to give rise to novel (reassorted) viruses with increased zoonotic potential.
In multiple stages of an epidemic, serological studies can assist in understanding of spread of infections. In this prospective, our work adds to the increasing consideration of using comprehensive serological methods to monitor virus prevalence and emergence.