Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/µL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

Research Progress of Porcine Reproductive and Respiratory Syndrome Virus NSP2 Protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) is globally prevalent and seriously harms the economic efficiency of pig farming. Because of its immunosuppression and high incidence of mutant recombination, PRRSV poses a great challenge for disease prevention and control. Nonstructural protein 2 (NSP2) is the most variable functional protein in the PRRSV genome and can generate NSP2N and NSP2TF variants due to programmed ribosomal frameshifts. These variants are broad and complex in function and play key roles in numerous aspects of viral protein maturation, viral particle assembly, regulation of immunity, autophagy, apoptosis, cell cycle and cell morphology. In this paper, we review the structural composition, programmed ribosomal frameshift and biological properties of NSP2 to facilitate basic research on PRRSV and to provide theoretical support for disease prevention and control and therapeutic drug development.

Pathogenicity and Immunogenicity of a Serially Passaged Attenuated Genotype 2c Porcine Epidemic Diarrhea Virus Cultured in Suspended Vero Cells.

In this study, one G2c-subtype strain of porcine epidemic diarrhea virus (PEDV) (SHXX1902 strain) was isolated from clinical samples in suspended Vero cells, which was different from the genotype of the commercial AJ1102 vaccine. As a result, we determined the pathogenicity of different passages' isolates (SHXX1902 strain) and compared the immunogenicity of G2c-subtype strain (SHXX1902 strain) with the commercial AJ1102 vaccine. The viral titer reached 107 50% tissue culture infectious dose (TCID50)/ml, which met the requirement for seed virus replication during vaccine development. Five-day-old piglets were orally infected with viruses from passages P5 and P35 to determine the pathogenicity and immunogenicity of different passages. Pregnant sows were immunized with inactivated SHXX1902-P5 or the commercial AJ1102 vaccine (first immunized with an attenuated vaccine and then boosted with an inactivated vaccine) to study the influence of the culture method on the immunogenicity of the strain. The median pig diarrhea dose (PDD50) and the median lethal dose (LD50) of the P5 virus were 102.00 and 102.84 TCID50/ml, respectively. All five piglets infected with the SHXX1902-P5 virus shed the virus 24 h after vaccination, whereas only two of the five piglets treated with the SHXX1902-P35 virus shed the virus 48 h after vaccination. The SHXX1902-P35 virus was partially attenuated in the 5-day-old piglets. Inactivated SHXX1902-P5 induced PEDV-specific immunoglobulin G (IgG) antibody responses equivalent to those induced by AJ1102 after infection in sow serum. However, the IgA titer induced by AJ1102 was much higher than that induced by inactivated SHXX1902-P5 since the boost immunization. On days 5 and 7 after farrowing, the IgA titers were similar among the immunized groups. Our study highlights that serial passage can lead to the attenuation of G2c-subtype strain. The immunogenicity of the inactivated strain was similar to the commercial vaccine. Our observation helped conceptualize appropriate study designs for the PEDV vaccine.

Phylogenetic analysis and evolutionary dynamics of H3N2 canine and feline influenza virus strains from 2006 to 2019.

H3N2 feline influenza virus (FIV) and canine influenza virus (CIV) are very common in cats and dogs. Due to the ability of the influenza virus to spread across hosts and frequent contact between pets and people, there exist huge public health problems. In this study, we collected H3N2 CIV and FIV genomes from 2006 to 2019 from NCBI and analyzed the evolutionary dynamics and molecular variation using a series of phylogenetic analysis methods. Results indicated that H3N2 FIVs were closely related to CIVs with high posterior probability and CIVs and FIVs have certain regional characteristics. However, compared with previous studies, the significance of geographical structure correlation decreased. Furthermore, we also found that the intrasubtypic reassortment between FIVs and CIVs were common during epidemics. The integrated analysis was also performed for different selection pressure acting on HA (566 codons), NA (469 codons), M1 (252 codons), and M2 (97 codons) proteins. One HA, two NA, three M1, and two M2 sites were found under positive selection. We subsequently performed the evolutionary dynamics of H3N2 CIV. The results indicated that the time of the most recent common ancestor of CIV H3N2 may have occurred earlier than indicated in a previous study. The Bayesian skyline plot analysis in this study showed the period of divergence of major H3N2 CIVs segments occurred between 2008 and 2010. Notably, according to our research, the PB1 has experienced two divergence periods (2006-2008 and 2009-2011).

Novel Method for Isolation of Porcine Epidemic Diarrhea Virus with the Use of Suspension Vero Cells and Immunogenicity Analysis.

In February and December of 2019, two large-scale outbreaks of diarrhea were observed in the same swine farm with 3,000 sows in Shanghai, China. We successfully isolated two porcine epidemic diarrhea virus (PEDV) isolates (strains shxx1902 and shxx1912 in February and December, respectively) from clinical samples in this farm using suspension Vero cells. A third PEDV strain (SH1302) tested positive in another farm of Shanghai, China, in 2013 and was also isolated using suspension Vero cells. The three isolates were better adapted to growth in adherent Vero cells through serial passages in the suspension Vero cells. The three isolated strains were detected positive by an immunofluorescence assay (IFA) and observed through electron microscopy. Phylogenetic analysis of the complete genomic sequence demonstrated that shxx1902 (the 5th passage) and shxx1912 (the 5th passage) clustered with a new GII subgroup (GII-c), which consisted of SINDEL strains from America (e.g., OH851), and their S gene belonged to GII-a. Both strains(the 35th passage) have incurred dramatic changes in their genomes compared with the 5th passage. The 5th and 35th passages of SH1302 belonged to the GI-b genotype. The anti-N protein antibody titer of the strain shxx1902 was elevated to the same level as the vaccine strain (CV777) in mice. The use of the suspension Vero cells to isolate and propagate PEDV provides an effective approach for studies of the epidemiological characteristics and vaccine development of this virus.

Influenza virus-like particles harboring H9N2 HA and NA proteins induce a protective immune response in chicken.

BACKGROUND: Avian influenza viruses represent a growing threat of an influenza pandemic. The co-circulation of multiple H9N2 genotypes over the past decade has been replaced by one predominant genotype-G57 genotype, which displays a changed antigenicity and improved adaptability in chickens. Effective H9N2 subtype avian influenza virus vaccines for poultry are urgently needed. OBJECTIVE: In this study, we constructed H9N2 subtype avian influenza virus-like particle (VLP) and evaluated its protective efficacy in specific pathogen-free (SPF) chickens to lay the foundation for developing an effective vaccine against influenza viruses. METHODS: Expression of influenza proteins in VLPs was confirmed by Western blot, hemagglutination inhibition (HI), and neuraminidase inhibition (NI). The morphology was observed by electron microscopy. A group of 15 three-week-old SPF chickens was divided into three subgroups of five chickens immunized with VLP, commercial vaccine, and PBS. Challenge study was performed to evaluate efficacy of VLP vaccine. RESULTS AND CONCLUSIONS: The hemagglutinin (HA) and neuraminidase (NA) proteins were co-expressed in the infected cells, self-assembled, and were released into the culture medium in the form of VLPs of diameter ~80 nm. The VLPs exhibited some functional characteristics of a full influenza virus, including hemagglutination and neuraminidase activity. In SPF chickens, the VLPs elicited serum antibodies specific for H9N2 and induced a higher HI titer (as detected by a homologous antigen) than did a commercial H9N2 vaccine (A/chicken/Shanghai/F/1998). Viral shedding from VLP vaccine subgroup was reduced compared with commercial vaccine subgroup and control subgroup.

Genotypic evolution and antigenicity of H9N2 influenza viruses in Shanghai, China.

H9N2 influenza viruses have been circulating in China since 1994, but a systematic investigation of H9N2 in Shanghai has not previously been undertaken. Here, using 14 viruses we isolated from poultry and pigs in Shanghai during 2002 and 2006-2014, together with the commercial vaccine A/chicken/Shanghai/F/1998 (Ck/SH/F/98), we analyzed the evolution of H9N2 influenza viruses in Shanghai and showed that all 14 isolates originated from Ck/SH/F/98 antigenically. We evaluated the immune protection efficiency of the vaccine. Our findings demonstrate that H9N2 viruses in Shanghai have undergone extensive reassortment. Various genotypes emerged in 2002, 2006 and 2007, while during 2009-2014 only one genotype was found. Four antigenic groups, A-D, could be identified among the 14 isolates and a variety of antigenically distinct H9N2-virus-derived avian influenza viruses (AIVs) circulated simultaneously in Shanghai during this period. Challenge experiments using vaccinated chickens indicated that the vaccine prevented shedding of antigenic group A and B viruses, but not those of the more recent groups C and D. Genetic analysis showed that compared to the vaccine strain, representative viruses of antigenic groups C and D possess greater numbers of amino acid substitutions in the hemagglutinin (HA) protein than viruses in antigenic groups A and B. Many of these substitutions are located in antigenic sites. Our results indicate that the persistence of H9N2 AIV in China might be due to incomplete vaccine protection and that the avian influenza vaccine should be regularly evaluated and updated to maintain optimal protection.

Genetic analysis of H3N2 avian influenza viruses isolated from live poultry markets and poultry slaughterhouses in Shanghai, China in 2013.

Five H3N2 avian influenza viruses (AIVs) were isolated from live poultry markets (LPMs) and poultry slaughterhouses in Shanghai, China in 2013. All viruses were characterized by whole-genome sequencing with subsequent genetic comparison and phylogenetic analysis. The hemagglutinin cleavage site of all viruses indicated that the five strains were low-pathogenic AIVs. Phylogenetic analysis of all eight viral genes showed that the five H3N2 viruses clustered in the Eurasian lineage of influenza viruses. The eight genes showed evidence of reassortment events between these H3 subtype viruses and other subtype viruses, especially H5 and H7 subtypes, probably in pigeons, domestic ducks, and wild birds. These findings emphasized the importance of AIV surveillance in LPMs and poultry slaughterhouses for understanding the genesis and emergence of novel reassortants with pandemic potential.

Epidemiological situation and genetic analysis of H7N9 influenza viruses in Shanghai in 2013.

The first reported human case of H7N9 influenza virus infection in Shanghai prompted a survey of local avian strains of influenza virus, involving the analysis of a large number of samples taken from poultry, wild birds, horses, pigs, dogs and mice. Seven instances of H7N9 virus infection were identified by real-time RT-PCR (1.47 % of samples), all in chickens sold in live-poultry markets. H7N9 antibody was not detected in serum samples collected from local poultry farms since 2006. The two H7N9 virus strains in the live-poultry markets and one H9N2 virus strain in the same market were genetically characterized. Resequencing of two of the seven isolates confirmed that they closely resembled H7N9 virus strains characterized elsewhere. Various strains co-exist in the same market, presenting a continuing risk of strain re-assortment. The closure of live-poultry markets has been an effective short-term means of minimizing human exposure to H7N9 virus.

Whole-genome analysis of porcine epidemic diarrhea virus (PEDV) from eastern China.

The complete genome sequence of a porcine epidemic diarrhea virus variant, strain SHQP/YM/2013, from China was determined and compared with those of other porcine epidemic diarrhea viruses. The full-length genome was 28,038 nucleotides (nt) in length without the poly (A) tail, and it was similar to that of other reported PEDV strains, with the characteristic gene order 5'-replicase (1a/1b) -S-ORF3-E-M-N-3'. Nucleotide sequence analysis based on individual virus genes indicated a close relationship between the S gene of SHQP/YM/2013 and those of the four Korean field strains from 2008-2009. Its ORF3 gene, however, fell into three groups. Recent prevalent Chinese PEDV field isolates were divided between group 1 and group 3, which suggests that the recent prevalent Chinese PEDV field isolates represent a new genotype that differs from the genotype that includes the vaccine strains. Based on phylogenetic analysis of the M gene, ORF3 gene and S gene, our study demonstrated that prevalent PEDV isolates in China may have originated from Korean strains. This report describes the complete genome sequence of SHQP/YM/2013, and the data will promote a better understanding of the molecular epidemiology and genetic diversity of PEDV field isolates in eastern China.

Epidemiological survey of porcine epidemic diarrhea virus in swine farms in Shanghai, China.

An epidemiological survey of porcine diarrheal disease prevalence between September 2011 and January 2012 revealed that porcine epidemic diarrhea virus (PEDV) contributed to outbreaks of diarrhea in pig farms in Shanghai, China. The distribution profile of 10 PEDV strains revealed three distinct genotypes coexisting in the same pig farm. Two of the ten field strains that were isolated exhibited a distinct evolution from the others. In addition to PEDV, other enteric pathogens, including porcine kobuvirus, porcine teschovirus and Lawsonia intracellularis, were identified.