RESUMEN

Recently, we identified the coxsackie and adenovirus receptor (CAR) as the entry receptor for rhesus enteric calicivirus (ReCV) isolate FT285 and demonstrated that co-expression of the CAR and the type B histo-blood group antigen (HBGA) is required to convert the resistant CHO cell line susceptible to infection. To address whether the CAR is also the functional entry receptor for other ReCV isolates and the requirement for specific HBGAs or other glycans, here we used a panel of recombinant CHO cell lines expressing the CAR and the type A, B, or H HBGAs alone or in combination. Infection studies with three diverse ReCV strains, the prototype GI.1 Tulane virus (TV), GI.2 ReCV-FT285, and GI.3 ReCV-FT7, identified that cell surface expression of the CAR is an absolute requirement for all three strains to promote susceptibility to infection, while the requirement for HBGAs varies among the strains. In addition to the CAR, ReCV-FT285 and TV require type A or B HBGAs for infection. In the absence of HBGAs, TV, but not Re-CV FT285, can also utilize sialic acids, while ReCV-FT7 infection is HBGA-independent and relies on CAR and sialic acid expression. In summary, we demonstrated strain-specific diversity of susceptibility requirements for ReCV infections and that CAR, type A and B HBGA, and sialic acid expression control susceptibility to infection with the three ReCV isolates studied. Our study also indicates that the correlation between in vitro HBGA binding and HBGAs required for infection is relatively high, but not absolute. This has direct implications for human noroviruses.IMPORTANCEHuman noroviruses (HuNoVs) are important enteric pathogens. The lack of a robust HuNoV cell culture system is a bottleneck for HuNoV cell culture-based studies. Often, cell culture-adapted caliciviruses that rapidly replicate in conventional cell lines and recapitulate biological features of HuNoVs are utilized as surrogates. Particularly, rhesus enteric caliciviruses (ReCVs) display remarkable similarities, including the primate host, clinical manifestation of gastroenteritis, genetic/antigenic diversity, and reliance on histo-blood group antigens (HBGAs) for attachment. While the HuNoV entry receptor(s) is unknown, the coxsackie and adenovirus receptor (CAR) has recently been identified as the ReCV entry receptor. Here, we identified the CAR, the type A and B HBGAs, and sialic acids as critical cell surface molecules controlling susceptibility to ReCV infections. The CAR is required for all ReCV isolates studied. However, the requirement for the different carbohydrate molecules varies among different ReCV strains. Our findings have direct implications for HuNoVs.

Asunto(s)

Infecciones por Caliciviridae , Caliciviridae , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Animales , Cricetinae , Humanos , Antígenos de Grupos Sanguíneos/metabolismo , Caliciviridae/fisiología , Infecciones por Caliciviridae/virología , Células CHO , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Intestino Delgado/virología , Ácido N-Acetilneuramínico/metabolismo , Norovirus/fisiología

RESUMEN

Recoviruses (rhesus enteric caliciviruses) are members of the Caliciviridae family. They are a valuable model for studying human caliciviruses such as noroviruses. It has been suggested that some recoviruses may infect humans, which necessitates detailed studies on the cell type tropism of recoviruses. For the recoviruses that have been cultured to date, successful growth has only been reported in monkey kidney cell lines, precluding their use to study virus interactions with human cells. We isolated and characterized a new recovirus, Recovirus Mo/TG30/2012, from monkey stool which grew efficiently in the monkey kidney cell line LLC-MK2. Notably, the virus can infect and replicate in several human cell lines derived from different organs. The ability to infect a human cell culture system with a recovirus expands our understanding of the potential for spillover to humans as well as increases the value of recoviruses as a model of human caliciviruses.

Asunto(s)

Infecciones por Caliciviridae , Caliciviridae , Norovirus , Virus ARN , Humanos , Caliciviridae/genética , Caliciviridae/metabolismo , Norovirus/genética , Línea Celular , Intestino Delgado

RESUMEN

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCV strains can be propagated to high titers in standard nonhuman primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells, which identified the coxsackievirus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that short interfering RNA, anti-human CAR (hCAR) monoclonal antibody RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9-targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection, and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate, caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays, we identified and validated the coxsackievirus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs both are necessary to convert a nonsusceptible cell line to being susceptible to ReCV infection. Follow-up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.

Asunto(s)

Infecciones por Caliciviridae/metabolismo , Receptores Virales/metabolismo , Replicación Viral/fisiología , Infecciones por Adenoviridae/metabolismo , Animales , Células CHO , Caliciviridae/metabolismo , Chlorocebus aethiops , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Infecciones por Coxsackievirus/metabolismo , Cricetulus , Gastroenteritis/virología , Intestino Delgado/inmunología , Macaca mulatta/inmunología , Modelos Biológicos , Norovirus/fisiología , Virus ARN/metabolismo , Receptores Virales/genética , Receptores Virales/fisiología , Células Vero , Acoplamiento Viral

RESUMEN

Besides noroviruses, the Caliciviridae family comprises four other accepted genera: Sapovirus, Lagovirus, Vesivirus, and Nebovirus. There are six new genera proposed: Recovirus, Valovirus, Bavovirus, Nacovirus, Minovirus, and Salovirus. All Caliciviridae have closely related genome structures, but are genetically and antigenically highly diverse and infect a wide range of mammalian host species including humans. Recombination in nature is not infrequent for most of the Caliciviridae, contributing to their diversity. Sapovirus infections cause diarrhoea in pigs, humans and other mammalian hosts. Lagovirus infections cause systemic haemorrhagic disease in rabbits and hares, and vesivirus infections lead to lung disease in cats, vesicular disease in swine, and exanthema and diseases of the reproductive system in large sea mammals. Neboviruses are an enteric pathogen of cattle, differing from bovine norovirus. At present, only a few selected caliciviruses can be propagated in cell culture (permanent cell lines or enteroids), and for most of the cultivatable caliciviruses helper virus-free, plasmid only-based reverse genetics systems have been established. The replication cycles of the caliciviruses are similar as far as they have been explored: viruses interact with a multitude of cell surface attachment factors (glycans) and co-receptors (proteins) for adsorption and penetration, use cellular membranes for the formation of replication complexes and have developed mechanisms to circumvent innate immune responses. Vaccines have been developed against lagoviruses and vesiviruses, and are under development against human noroviruses.

Asunto(s)

Infecciones por Caliciviridae/veterinaria , Caliciviridae/clasificación , Animales , Caliciviridae/patogenicidad , Infecciones por Caliciviridae/virología , Genoma Viral , Humanos , Norovirus , Filogenia , Análisis de Secuencia de ADN

RESUMEN

Human noroviruses (NoV) are associated with large proportion of non-bacterial diarrhea outbreaks together with > 50% of food-associated diarrheas. The function of histo-blood group antigens (HBGAs) in pathogenesis of virus infection was implicated. Until recently however, due to lack of a robust animal and in vitro models of human NoV infection, only the partial knowledge concerning the virus pathogenesis (receptor, co-receptor and target cell) and absence of viable vaccine candidates were the frequently referenced attributes of this acute diarrheal illness. Recently, a novel group of enteric caliciviruses (CV) of rhesus macaque host origin was discovered and described. The new genus within the family Caliciviridae was identified: Rhesus Enteric CV, i.e., "Recovirus" (ReCV). ReCVs are genetically and biologically close relatives of human NoVs, exhibit similar genetic and biological features and are capable of being propagated in cell culture. ReCVs cause symptomatic disease (diarrhea and fever) in experimentally inoculated macaques. Formulation and evaluation of efficient NoV vaccine might take several years. As suggested by recent studies, inhibition of HBGAs or HBGA-based antivirals could meanwhile be exploited as vaccine alternatives. The purpose of this minireview is to provide the guidance in respect to newly available primate model of enteric CV infection and its similarities with human NoV in utilizing the HBGAs as potential virus co-receptors to indirectly address the unresolved questions of NoV pathogenesis and immunity.