The long-term research goals in Dr. Li’s laboratory are to understand the molecular mechanisms of RNA virus entry, replication, and pathogenesis, and to facilitate the rational design and development of new vaccines, anti-viral drugs, and prevention strategies. Currently, Dr. Li’s laboratory is studying a number of RNA viruses including human metapneumovirus (hMPV), human respiratory syncytial virus (RSV), human norovirus, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and SARS-CoV-2.
Research interest I: Human metapneumovirus (hMPV) and human respiratory syncytial virus (RSV).
Pneumoviridae is a new virus family in the order Mononegavirales. It was created in 2016 by elevating the paramyxoviral subfamily Pneumovirinae to family status. The Pneumoviridae family includes two medically important pathogens, human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) which are the no.1 and 2 causative agents of acute viral respiratory tract infections in infants, young children, the elderly, and immunocompromised individuals. Despite the enormous economic loss and emotional burden these viruses cause, no vaccines or anti-viral drugs are currently available. Development of such agents requires a better understanding of all aspects of their life cycle.
Project 1: Understand the replication and gene expression of metapneumoviruses. All viruses utilize host cell machinery to synthesize their own genetic materials. Upon entry into cells, the virus has to initiate replication and gene expression. Pneumoviruses use a unique strategy for replication and gene expression. The active template for pneumovirus replication is not the naked RNA genome but the protein and RNA complex. Viral genomic RNA is completely encapsidated by the nucleocapsid (N) protein to form an N-RNA complex. During RNA synthesis, the N-RNA template is recognized by viral RNA-dependent RNA polymerase (RdRp) that carries out two distinct processes: (i) transcription to yield capped, methylated and polyadenylated messenger RNAs and (ii) replication to yield full-length antigenomic and subsequently genomic RNA.
Our lab uses hMPV as models to understand the mechanisms by which the RdRp controls these two processes: replication and transcription. We will focus on the two major components of RdRp, the large protein catalytic subunit (L) and the cofactor phosphoprotein (P). We will use biochemical and genetic approaches to define functional domains in the RdRd that regulate replication and transcription. Disruption of these functional domains in the RdRp could potentially limit virus replication and thus attenuate the virus, or alternatively could be lethal to the virus. Therefore, a better understanding of replication and transcription control would facilitate the rational design and development of new vaccines and anti-viral drugs against these viruses.
Project 2: Understand the mechanism of internal RNA methylation in pneumoviruses. We recently discovered that pneumovirus RNAs are methylated at internal adenosine residues to form N6-methyladenosine (m6A) by host m6A methyltransferase complex. Although m6A methylation has been discovered in viral RNA in early 1970s, the biological function of m6A in the virus life cycle, pathogenesis, and immunity has been a mystery for four decades. Our laboratory is interested in determining the roles of internal m6A methylation in pneumovirus replication and pathogenesis in vivo; and to define mechanism(s) by which m6A methylation modulate pneumovirus life cycle.
Project 3: Develop new vaccine candidates for hMPV and RSV. Messenger RNAs (mRNAs) of pneumoviruses typically possess guanine-N-7 (G-N-7) and ribose 2′-O (2′-O) methylation. It is known that G-N-7 methylation is essential for mRNA stability as well as efficient translation whereas ribose 2′-O methylation provides a molecular signature for the discrimination of self and nonself mRNA by innate immunity. Our laboratory is interested in developing mRNA cap methyltransferase-defective recombinant viruses as novel live vaccine candidates for RSV and hMPV. All these vaccine candidates will be tested in in primary well differentiated human airway epithelial (HAE) cultures, cotton rats, and nonhuman primates.
Research interest II: Human norovirus and animal caliciviruses.
The Caliciviridae family includes a number of significant enteric viruses that cause gastroenteritis in humans and animals. Examples of these viruses include human norovirus, human sapovirus, porcine norovirus, and the newly discovered primate calicivirus (Tulane virus). Human norovirus is the leading cause of nonbacterial gastroenteritis worldwide, contributing to over 95% of all non-bacterial acute gastroenteritis each year, and more than 60% of all foodborne illnesses reported annually. However, human norovirus remains difficult to study because it cannot be grown in cell culture and it lacks a small animal model. Currently, there is no vaccine or antiviral drug against this virus.
Project 1: Understand the pathogenesis of human norovirus and primate calicivirus in a gnotobiotic pig model.
Recently, we found that Tulane virus, an enteric cultivable primate calicivirus that is genetically similar to human norovirus, replicates in gnotobiotic pigs. Similar to human norovirus, Tulane virus causes diarrhea and fecal viral shedding, and histological changes in pig intestines. Using gnotobiotic pigs as a model, we aim to understand the pathological and immunological aspects of human norovirus and primate calicivirus in vivo.
Project 2: Develop new vaccine candidates against human norovirus and other noncultivable caliciviruses. To date, there are no vaccines or anti-viral therapies for human norovirus. This is due in major part to the fact that human norovirus cannot be grown in cell culture. Generally, a live attenuated vaccine stimulates strong systemic immunity and provides durable protection. However, such a vaccine is not realistic for viruses that cannot grow in cell culture. In this situation, a vectored vaccine may be ideal. We are interested in developing non-segmented negative-strand RNA viruses (such as vesicular stomatitis virus, VSV) as vectors to deliver human norovirus virus-like particles (VLPs) to be used as a candidate live vaccine for human norovirus. Using a gnotobiotic pig model, we will examine the protective immune response against human norovirus infection using these vaccine candidates. VSV offers a number of advantages, such as safety, genetic stability, efficient expression of foreign genes, and induction of systemic immune responses. This recombinant system will provide a new avenue for the development of vaccines for non-cultivable caliciviruses
Research Interest III: Porcine coronaviruses and SARS-CoV-2.
In May of 2013, porcine epidemic diarrhea virus (PEDV) suddenly emerged in the United States. Today, at least 30 states have reported piglet diarrheal disease and deaths attributable to PEDV infection. In February of 2014, another porcine coronavirus, porcine deltacoronavirus (PDCoV) emerged in Ohio and Indiana and subsequently spread rapidly across the United States and Canada, causing significant economic losses. Both PEDV and PDCoV replicate in small intestinal enterocytes causing life-threatening acute enteric disease in suckling piglets that is characterized by profuse watery diarrhea, emesis and resultant dehydration. Morbidity rates are high (80-100%) as are mortality rates (50-90%). Both PDCoV and PEDV have been declared as “reportable diseases” to the USDA and Canada. Currently, there is no vaccine or treatment for PEDV and PDCoV. Dr. Li’s laboratory recently isolated a number of PEDV and PDCoV Ohio strains, and successfully adapted these isolates to tissue culture. We are interested in understanding the host tropism, pathogenesis, and vaccines against PEDV and PDCoV.
In response to SARS-CoV-2 outbreak in 2019, our laboratory quickly developed several SARS-CoV-2 vaccine candidates using non-segmented negative-sense RNA viruses as the vectors. Among them, the measles virus-based SARS-CoV-2 vaccine candidates have been published in PNAS in 2021 and licensed to Biological E Limited. We are currently testing other SARS-CoV-2 vaccine candidates in several animal models. The lab is also interested in RNA modifications in SARS-CoV-2 and development of novel therapeutics against SARS-CoV-2 infection.