Serial Passaging of the Human Rotavirus CDC-9 Strain in Cell Culture Leads to Attenuation: Characterization from In Vitro and In Vivo Studies.

Live oral rotavirus vaccines have been developed by serial passaging in cell culture and found to be safe in infants. However, mechanisms for the adaptation and attenuation of rotavirus vaccines are not fully understood. We prepared a human rotavirus vaccine strain, CDC-9 (G1P[8]), which when grown in MA104 cells to passage 11 or 12 (P11/P12) had no nucleotide or amino acid sequence changes from the original virus in stool. Upon adaptation and passages in Vero cells, the strain underwent five amino acid changes at P28 and one additional change at P44/P45 in the VP4 gene. We performed virologic, immunological, and pathogenic characterization of wild-type CDC-9 virus at P11/P12 and its two mutants at P28 or P44/P45 using in vitro and in vivo model systems. We found that mutants CDC-9 P28 and P44 induced upregulated expression of immunomodulatory cytokines. On the other hand, the two mutant viruses induced lower STAT1 phosphorylation and grew to 2-log-higher titers than wild-type virus in human Caco-2 cells and simian Vero cells. In neonatal rats, CDC-9 P45 showed reduced rotavirus shedding in fecal specimens and did not induce diarrhea compared to wild-type virus and modulated cytokine responses comparably to Rotarix infection. These findings indicate that mutant CDC-9 is attenuated and safe. Our study is the first to provide insight into the possible mechanisms of human rotavirus adaptation and attenuation and supports ongoing efforts to develop CDC-9 as a new generation of rotavirus vaccine for live oral or parenteral administration.IMPORTANCE Mechanisms for in vitro adaptation and in vivo attenuation of human rotavirus vaccines are not known. The present study is the first to comprehensively compare the in vitro growth characteristics, virulence, and host response of a wild-type and an attenuated human rotavirus strain, CDC-9, in Caco-2 cells and neonatal rats. Our study identifies critical sequence changes in the genome that render human rotavirus adapted to growth to high levels in Vero cells and attenuated and safe in neonatal rats; thus, the study supports clinical development of CDC-9 for oral or parenteral vaccination in children.

Genetic-Code-Expansion Strategies for Vaccine Development.

By providing long-term protection against infectious diseases, vaccinations have significantly reduced death and morbidity worldwide. In the 21st century, (bio)technological advances have paved the way for developing prophylactic vaccines that are safer and more effective as well as enabling the use of vaccines as therapeutics to treat human diseases. Here, we provide a focused review of the utility of genetic code expansion as an emerging tool for the development of vaccines. Specifically, we discuss how the incorporation of immunogenic noncanonical amino acids can aid in eliciting immune responses against adverse self-proteins and highlight the potential of an expanded genetic code for the construction of replication-incompetent viruses. We close the review by discussing the future prospects and remaining challenges for the application of these approaches in the development of both prophylactic and therapeutic vaccines in the near future.

S2 subunit plays a critical role in pathogenesis of TW-like avian coronavirus infectious bronchitis virus.

To investigate the critical role of the S gene in determining pathogenesis of TW-like avian infectious bronchitis virus (IBV), we generated two recombinant IBVs (rGDaGD-S1 and rGDaGD-S2) by replacing either the S1 or S2 region of GD strain with the corresponding regions from an attenuated vaccine candidate aGD strain. The virulence and pathogenicity of these recombinant viruses was assessed both in vitro and in vivo. Our results indicated the mutations in the S2 region led to decreased virulence, as evidenced by reduced virus replication in embryonated chicken eggs and chicken embryonic kidney cells as well as observed clinical symptoms, gross lesions, microscopic lesions, tracheal ciliary activity, and viral distribution in SPF chickens challenged with recombinant IBVs. These findings highlight that the S2 subunit is a key determinant of TW-like IBV pathogenicity. Our study established a foundation for future investigations into the molecular mechanisms underlying IBV virulence.

Characterisation of a Live-Attenuated Rabies Virus Expressing a Secreted scFv for the Treatment of Rabies.

Rabies virus (RABV) causes possibly the oldest disease and is responsible for an estimated >59,000 human fatalities/year. Post exposure prophylaxis (PEP), the administration of vaccine and rabies immunoglobulin, is a highly effective tool which is frequently unavailable in RABV endemic areas. Furthermore, due to the constraints of the blood-brain barrier, current PEP regimes are ineffective after the onset of clinical symptoms which invariably result in death. To circumvent this barrier, a live-attenuated recombinant RABV expressing a highly RABV-neutralising scFv antibody (62-71-3) linked to the fluorescent marker mCherry was designed. Once rescued, the resulting construct (named RABV-62scFv) was grown to high titres, its growth and cellular dissemination kinetics characterised, and the functionality of the recombinant 62-71-3 scFv assessed. Encouraging scFv production and subsequent virus neutralisation results demonstrate the potential for development of a therapeutic live-attenuated virus-based post-infection treatment (PIT) for RABV infection.

Vaccine Candidates against Arenavirus Infections.

The viral family Arenaviridae contains several members that cause severe, and often lethal, diseases in humans. Several highly pathogenic arenaviruses are classified as Risk Group 4 agents and must be handled in the highest biological containment facility, biosafety level-4 (BSL-4). Vaccines and treatments are very limited for these pathogens. The development of vaccines is crucial for the establishment of countermeasures against highly pathogenic arenavirus infections. While several vaccine candidates have been investigated, there are currently no approved vaccines for arenavirus infection except for Candid#1, a live-attenuated Junin virus vaccine only licensed in Argentina. Current platforms under investigation for use include live-attenuated vaccines, recombinant virus-based vaccines, and recombinant proteins. We summarize here the recent updates of vaccine candidates against arenavirus infections.

Failure to experimentally infect 10 days-old piglets with a cell culture-propagated infectious stock of a classical genotype 1a porcine epidemic diarrhea virus.

Introduction: Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs of all ages. PEDV can be grouped into G1 (classical strains) and G2 (variant strains) based on sequence differences in the spike gene. Although several pathogenesis studies using contemporary strains of PEDV have been conducted to date, there is limited information on the pathogenesis of historical PEDV strains in contemporary pigs. This study aimed to investigate the clinical disease course of 10 days-old pigs infected with a classical European G1a PEDV strain from the 1980s which was last passaged in pigs in 1994. Methods: Sequencing results confirmed that the virus inoculum was a PEDV strain closely related to the prototype CV777 strain. The PEDV stock was serially passaged three times in Vero cells, and the P3 infectious virus stock was used to inoculate the pigs. A total of 40 pigs were inoculated using the oral route. Results: Pigs showed no enteric disease signs, and PEDV shedding was not detected for 44 days post-inoculation (dpi). At necropsy at 3 (5 pigs) or 7 dpi (5 pigs), no lesions were observed in intestinal sections, which were negative for PEDV antigen by immunohistochemistry. In addition, no IgG or IgA PEDV-specific antibodies in serum or fecal samples for 35 dpi further indicates a lack of infection. Titration of the leftover thawed and refrozen PEDV virus stock inoculum showed that the virus stock retained its infectivity in Vero cell culture and the porcine small intestine enterocytes cell line IPEC-J2. Discussion: The reasons for the loss of infectivity in pigs are unknown. In conclusion, we showed that a classical G1a PEDV strain successfully propagated in cell cultures could not orally infect 40 piglets.

NS1 and PA-X of H1N1/09 influenza virus act in a concerted manner to manipulate the innate immune response of porcine respiratory epithelial cells.

Live-attenuated influenza A viruses (LAIV) may be superior to inactivated or subunit vaccines since they can be administered via mucosal routes to induce local immunity in the respiratory tract. In addition, LAIV are expected to trigger stronger T-cell responses that may protect against a broader range of antigen-drifted viruses. However, the development of LAIV is challenging since a proper balance between immunogenicity and safety has to be reached. In this study, we took advantage of reverse genetics to generate three LAIV based on the pandemic H1N1 2009 (pH1N1/09) virus strain: ΔPA-X, which is defective in the synthesis of the accessory PA-X protein, NS1(1-126) lacking 93 amino acids at the C-terminus of the NS1 protein, and a combination of both. Characterization of these recombinant viruses using a novel porcine bronchiolar epithelial cell line (T3) revealed that the ΔPA-X mutant replicated similar to wild type (WT) virus. However, in contrast to the parental virus the ΔPA-X mutant allowed transcription of genes involved in cell cycle progression and limits apoptosis. The NS1(1-126) mutant also replicated comparable to WT virus, but triggered the release of type I and III IFN and several chemokines and cytokines. Surprisingly, only the NS1(1-126)/ΔPA-X double mutant was significantly attenuated on T3 cells, and this was associated with enhanced transcription of genes of the innate immune system and complete absence of apoptosis induction. In conclusion, these findings indicate that NS1 and PA-X act in a concerted manner to manipulate the host cell response, which may help to develop swine LAIV vaccine with a more favorable balance of safety and immunogenicity.

An attenuated TW-like infectious bronchitis virus strain has potential to become a candidate vaccine and S gene is responsible for its attenuation.

TW-like infectious bronchitis virus (IBV) with high pathogenicity is becoming the predominant IBV type circulating in China. To develop vaccines against TW-like IBV strains and investigate the critical genes associated with their virulence, GD strain was attenuated by 140 serial passages in specific-pathogen-free embryonated eggs and the safety and efficacy of the attenuated GD strain (aGD) were examined. The genome sequences of GD and aGD were also compared and the effects of mutations in the S gene were observed. The results revealed that aGD strain showed no obvious pathogenicity with superior protective efficacy against TW-like and QX-like virulent IBV strains. The genomes of strains aGD and GD shared high similarity (99.87 %) and most of the mutations occurred in S gene. Recombinant IBV strain rGDaGD-S, in which the S gene was replaced with the corresponding regions from aGD, showed decreased pathogenicity compared with its parental strain. In conclusion, attenuated TW-like IBV strain aGD is a potential vaccine candidate and the S gene is responsible for its attenuation. Our research has laid the foundation for future exploration of the attenuating molecular mechanism of IBV.

Evolutionary Patterns of Codon Usage in Major Lineages of Porcine Reproductive and Respiratory Syndrome Virus in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is economically important and characterized by its extensive variation. The codon usage patterns and their influence on viral evolution and host adaptation among different PRRSV strains remain largely unknown. Here, the codon usage of ORF5 genes from lineages 1, 3, 5, and 8, and MLV strains of type 2 PRRSV in China was analyzed. A compositional property analysis of ORF5 genes revealed that nucleotide C is most frequently used at the third position of codons, accompanied by rich GC3s. The effective number of codon (ENC) and codon pair bias (CPB) values indicate that all ORF5 genes have low codon bias and the differences in CPB scores among four lineages are almost not significant. When compared with host codon usage patterns, lineage 1 strains show higher CAI and SiD values, with a high similarity to pig, which might relate to its predominant epidemic propensity in the field. The CAI, RCDI, and SiD values of ORF5 genes from different passages of MLV JXA1R indicate no relation between attenuation and CPB or codon adaptation decrease during serial passage on non-host cells. These findings provide a novel way of understanding the PRRSV's evolution, related to viral survival, host adaptation, and virulence.

mSphere of Influence: Experimental Evolution of RNA Viruses.

Gonzalo Moratorio works in the field of experimental evolution of viruses. In this mSphere of Influence article, he reflects on how the papers "Virus attenuation by genome-scale changes in codon pair bias" by Coleman et al. (Science 320:1784-1787, 2008, https://doi.org/10.1126/science.1155761) and "Codon usage determines the mutational robustness, evolutionary capacity, and virulence of an RNA virus" by Lauring et al. (Cell Host Microbe 12:623-632, 2012, https://doi.org/10.1016/j.chom.2012.10.008) made an impact on his thinking about how to employ synthetic biology to study experimental evolution of viruses.

Comparative Genomic Analysis of Classical and Variant Virulent Parental/Attenuated Strains of Porcine Epidemic Diarrhea Virus.

Since 2010, the variant porcine epidemic diarrhea virus (PEDV) has been the etiological agent responsible for the outbreak of porcine epidemic diarrhea (PED) worldwide. In this study, a variant PEDV strain YN1 was isolated, serially propagated on the Vero cells and was characterized for 200 passages. To better elucidate the molecular basis of Vero cell adaptation of variant PEDV strains, we sequenced, compared, and analyzed the full-genome sequences of parental YN1 and passages 15, 30, 60, 90, 144, and 200. The results showed that the variations increased with the viral passage. The nucleotides sequences of non-structural protein (NSP)2, NSP4-7, NSP10, NSP12 and NSP13 genes did not change during the Vero cell adaptation process. After comparison of the variation characteristic of classical, variant virulent/attenuated strains, it was found that attenuation of PEDV virus was associated with 9-26 amino acid (aa) changes in open reading frames (ORF) 1a/b and S protein, early termination in ORF3, 1-3 aa changes in E, M and N protein and some nucleotide sequences' synonymous mutations. The aa deletion at about 144 aa of S protein could be the attenuation marker for the PEDV. The pig study showed that the early termination in ORF3 was more important for virus cell adaptation than virus attenuation.