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The intricate connection between the gut and the brain involves multiple routes. Several viral families begin their infection cycle in the intestinal tract. However, amongst the long list of viral intestinal pathogens, picornaviruses, and astroviruses stand out for their ability to transition from the intestinal epithelia to central or peripheral nervous system cells. In immunocompromised, neonates and young children, these viral infections can manifest as severe diseases, such as encephalitis, meningitis, and acute flaccid paralysis. What confers this remarkable plasticity and makes them efficient in infecting cells of the gut and the brain axes? Here, we review the current understanding of the virus infection along the gut-brain axis for some enteric viruses and discuss the molecular mechanisms of their attenuation.

Porcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172–554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.

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.

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.

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.

The research conducted in this preclinical study assesses QazCovid-live, a live attenuated COVID-19 vaccine created in Kazakhstan, by conducting preclinical evaluations of safety, immunogenicity, and allergenicity in various animal models, including mice, rats, hamsters, and guinea pigs. The vaccine, developed by attenuating SARS-CoV-2 via numerous Vero cell passages, had no significant adverse effects in acute and subacute toxicity assessments, even at elevated dosages. Allergenicity testing indicated the absence of both immediate and delayed hypersensitivity reactions. Immunogenicity evaluations revealed strong virus-neutralizing antibody responses, especially following intranasal and intratracheal delivery. Studies on reversibility and transmission further validated the vaccine’s stability and non-pathogenicity. The data indicate that QazCovid-live is safe, immunogenic, and prepared for clinical trials, presenting a potential strategy for COVID-19 prevention.

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.

Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.

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.

IntroductionPorcine 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.MethodsSequencing 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.ResultsPigs 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.DiscussionThe 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.