Coinfection caused by various genotypes of porcine epidemic diarrhea virus (PEDV) is a new disease situation. We previously reported the coexistence of PEDV strains containing different ORF3 genotypes in China. In this study, the PEDV strains 17GXCZ-1ORF3d and 17GXCZ-1ORF3c were isolated and plaque-purified from the same piglet, which had a natural large deletion at the 172–554 bp position of the ORF3 gene or possessed a complete ORF3 gene, respectively. Meanwhile, 17GXCZ-1ORF3d had >99% nt identity with 17GXCZ-1ORF3c in the 5′UTR, ORF1a/1b, S, E, M, N and 3′UTR regions but only demonstrated low nucleotide identities (80.5%) in the ORF3 gene. To elucidate the pathogenicity, 7-day-old piglets were infected. Piglets infected with these two PEDV strains exhibited severe clinical signs and shed the virus at the highest level within 96 hpi. Compared with the piglets inoculated with the 17GXCZ-1ORF3c strain, the piglets inoculated with the 17GXCZ-1ORF3d strain had higher mortality rates (75% vs. 50%), an earlier onset of clinical signs with a significantly higher diarrhea score, lower VH:CD ratios and a higher percentage of PEDV-positive enterocytes. This study is the first to report PEDV coinfections with different ORF3 genotypes, and a PEDV strain with a large deletion in the ORF3 gene might have the advantage of a potential genetic marker, which would be useful during vaccine development.

Live attenuated vaccines are critical in the control of avian infectious bronchitis. It is necessary to know the protection conferred by commonly used commercial live vaccines. In this study, specific pathogen-free chicks were vaccinated with the commercial live vaccines H120, 4/91 and LDT3-A. Blood samples were collected at weekly intervals for the detection of IBV-specific antibodies and quantification of CD4+ and CD8+ T lymphocytes. At 21 days post-inoculation the vaccinated birds were challenged with the IBV prevalent local strains GX-YL5, GX-GL11079 and GX-NN09032, respectively. Trachea and kidney samples were collected at 5 days post-challenge for the detection of the virus. The results showed that the H120 group exhibited medium antibody levels, the lowest percentages of CD4+, CD8+ T lymphocytes and the highest viral loads. The 4/91 group showed the lowest antibody levels, but the highest percentages of CD4+, CD8+ T lymphocytes and the lowest viral loads. The LDT3-A group showed the highest antibody levels, the medium percentages of CD4+, CD8+ T lymphocytes and the medium viral loads. The protection rates of H120, 4/91 and LDT3-A groups were 41.7–58.3%, 75.0–83.7% and 66.7–75.0%, respectively. The present study demonstrated that the vaccines H120, 4/91 and LDT3-A could stimulate the immunized chicks to produce different levels of humoral and cellular immunity to resist the infection of IBV, but couldn’t provide complete protection against the prevalent local strains of IBV in southern China. Also, the vaccine 4/91 offered the best immune protection among the three vaccines.

Porcine epidemic diarrhea virus (PEDV) is one of the major enteric pathogens, causing severe enteric disease, resulting in enormous economic losses. The ORF3 gene encodes an accessory protein which is related to the infectivity and virulence of PEDV. In this study, 33 PEDV positive field samples were collected from Guangxi, from 2017 to 2019, and the genetic diversity of ORF3 was investigated. Thirty-eight strains of ORF3 were obtained, and these were composed of five strains of ORF3 named Guangxi naturally truncated strains that were 293 bp in length, with continuous deletions from 172 to 554 bp. The Guangxi naturally truncated strains encoded a truncated protein of 89 amino acids, which had clustered into a new group referred to as Group 3, and these might be involved in the variations of virulence. Three genotypes (G1-1 subgroup, G1-3 subgroup, and Group 3) existed simultaneously in Guangxi based on the genetic and evolutionary analysis of the ORF3 gene. The sequence information in the current study will hopefully facilitate the establishment of a diagnostic method that can differentiate the PEDV field stains. Continued surveillance will be useful for monitoring PEDV transmission. Differentiation of the ORF3 genes in PEDV field strains can help us to choose an appropriate PEDV vaccine candidate in the future and prevent outbreaks of PED more effectively.