Expression of dysregulated miRNA in vivo in DF-1 cells during the course of subgroup J avian leukosis virus infection.

Aberrant expression of microRNAs (miRNAs) is known to be involved in cancer progression caused by subgroup J avian leukosis virus (ALV-J) in liver tissues. To advance our understanding of the related pathological mechanisms and virus-host interactions, seven previously reported miRNAs were selected for a comparative analysis of miRNA expression between infected and uninfected DF-1 cells, including six miRNAs related to tumorigenesis (let-7b/7i, miR-221/222, miR-125b, miR-375 and miR-2127. The results showed that six of the seven miRNAs except gga-miR-375 were upregulated in cells infected with NX0101 (caused myeloma (ML)) and GD1109 (caused hemangioma (HE)) at 1 h post infection. On day 2 post-infection, all seven miRNAs were upregulated in infected DF-1 cells. On day 6 post-infection, gga-let-7b, gga-miR-125b, and gga-miR-375 were downregulated whereas gga-miR-221 and gga-miR-222 were upregulated in DF-1 cells infected with the two ALV-J strains of different phenotypes. However, expression of gga-let-7i was reduced in DF-1 cells infected with NX0101 and was increased in those infected with GD1109; gga-miR-2127 expression showed no significant difference between infected and uninfected cells. This study is the first to report the changes in the miRNA expression levels in DF-1 cells during the course of ALV-J infection, and suggests a relationship between its pathological mechanisms and miRNAs.

Novel polymerase spiral reaction assay for the visible molecular detection of porcine circovirus type 3.

BACKGROUND: Porcine circovirus type 3 (PCV3) is a newly emerging circovirus that might be associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and cardiac and multisystemic inflammation. To aid the prevention and control of the infectious disease caused by PCV3, we developed a novel isothermal amplification assay using polymerase spiral reaction (PSR), which allows the visual detection of preserved strains and clinical samples. RESULTS: This assay precisely amplified the PCV3 genome with the use of a water bath at 62 °C for 50 min. The detection limit was found to be 1.13 × 102 copies/µL by gel electrophoresis or with the use of a visible dye (an indicator comprising phenol red and cresol red). No cross-reaction with other porcine infectious viruses was observed. The detection results for 23 PCV3-positive samples by PSR were in accordance with loop-mediated isothermal amplification (LAMP) assay. The detection rate of the PSR assay for PCV3 positivity of clinical samples was 68/97, which was higher than LAMP assay (67/97). CONCLUSIONS: These results indicated that the PSR assay provides an accurate and rapid method for the detection of PCV3 with high sensitivity and specificity. It is particularly suited for use in a simple laboratory setting without a thermal cycler or gel electrophoresis equipment.

Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a major etiological agent of porcine epidemic diarrhea around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The aim of this study was to establish a novel reverse transcription polymerase spiral reaction (RT-PSR) assay for the rapid detection of porcine epidemic diarrhea virus (PEDV). For the assay, a specific RT-PSR primer pair was designed against a conserved region in PEDV ORF3. RESULTS: The RT-PSR was optimized, and PEDV could be detected after a 50 min incubation at 62 °C, in addition to the 15 min required for reverse transcription. No cross-reaction with other porcine infectious viruses was observed. This new method for PEDV detection was 10 times more sensitive than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. The positive rates for 65 clinical samples using the new RT-PSR assay and the conventional RT-PCR assay were 58.46% (38/65) and 53.84% (35/65), respectively. In the RT-PSR assay, the addition of a mixture of dyes allowed a positive reaction to be directly observed by the naked eye. CONCLUSIONS: These results indicate that this RT-PSR assay is capable of accurately detecting PEDV, and has the advantages of high specificity and sensitivity for the detection of PEDV.

Xanthophyll supplementation regulates carotenoid and retinoid metabolism in hens and chicks.

This study investigated the effects of xanthophylls (containing 40% lutein and 60% zeaxanthin; Juyuan Biochemical Co., Ltd., GuangZhou, China) on gene expression associated with carotenoid cleavage enzymes (ß-carotene 15, 15'-monooxygenase, BCMO1; and ß-carotene 9', 10'-dioxygenase, BCDO2) and retinoid metabolism (lecithin:retinol acyl transferase (LRAT) and STRA6) of breeding hens and chicks. In experiment 1, 432 hens were divided into 3 groups and fed diets supplemented with zero (as the control group), 20, or 40 mg/kg xanthophyll. The liver, duodenum, jejunum, and ileum were sampled at d 35 of the trial. Results showed that 40 mg/kg xanthophyll supplementation increased BCDO2 mRNA in the liver, duodenum, and jejunum; LRAT mRNA in the jejunum; and STRA6 mRNA in the liver, while it decreased LRAT mRNA in the liver. Experiment 2 was a 2 × 2 factorial design. Male chicks hatched from a zero or 40 mg/kg xanthophyll diet of hens were fed a diet containing either zero or 40 mg/kg xanthophylls. The liver, duodenum, jejunum, and ileum were sampled at zero, 7, 14, and 21 d after hatching. Results showed that in ovo xanthophyll modulated carotenoid and retinoid metabolism mainly within one wk after hatching. The maternal effects gradually vanished and dietary effects began to work one to 2 wk after hatching. Dietary xanthophyll regulated carotenoid and retinoid metabolism mainly from 2 wk onward. The xanthophyll regulation of carotenoid and retinoid metabolism also revealed strong tissue specificity. In conclusion, xanthophyll supplementation could modulate carotenoid and retinoid metabolism in different tissues of hens and chicks.

Transcriptome-wide N6-methyladenosine modification and microRNA jointly regulate the infection of avian leukosis virus subgroup J in vitro.

N6-methyladenosine (m6A) methylation in transcripts has been suggested to influence tumorigenesis in liver tumors caused by the avian leukosis virus subgroup J (ALV-J). However, m6A modifications during ALV-J infection in vitro remain unclear. Herein, we performed m6A and RNA sequencing in ALV-J-infected chicken fibroblasts (DF-1). A total of 51 differentially expressed genes containing differentially methylated peaks were identified, which were markedly enriched in microRNAs (miRNAs) in cancer cells as well as apoptosis, mitophagy and autophagy, RNA degradation, and Hippo and MAPK signaling pathways. Correlation analysis indicated that YTHDC1 (m6A-reader gene) plays a key role in m6A modulation during ALV-J infection. The env gene of ALV-J harbored the strongest peak, and untranslated regions and long terminal repeats also contained peaks of different degrees. To the best of our knowledge, this is the first thorough analysis of m6A patterns in ALV-J-infected DF-1 cells. Combined with miRNA profiles, this study provides a useful basis for future research into the key pathways of ALV-J infection associated with m6A alteration.

Conservation and distribution of the DRACH motif for potential m6A sites in avian leukosis virus subgroup J.

N6-methyladenosine (m6A) methylation is an internal post-transcriptional modification that has been linked to viral multiplication and pathogenicity. To elucidate the conservation patterns of potential 5'-DRACH-3' motifs in avian leukosis virus subgroup J (ALV-J), 149 ALV-J strains (139 isolates from China; ALV-J prototype HPRS-103 from the UK; and 9 strains from the USA, Russia, India, and Pakistan) available in GenBank before December 2023 were retrieved. According to the prediction results of the SRAMP web-server, these ALV-J genomes contained potential DRACH motifs, with the total number ranging from 43 to 64, which were not determined based on the isolation region and time. Conservative analysis suggested that 37 motifs exhibited a conservation of >80%, including 17 motifs with a grading above "high confidence." Although these motifs were distributed in the U5 region of LTRs and major coding regions, they were enriched in the coding regions of p27, p68, p32, and gp85. The most common m6A-motif sequence of the DRACH motif in the ALV-J genome was GGACU. The RNA secondary structure of each conserved motif predicted by SRAMP and RNAstructure web-server was mainly of two types-A-U pair (21/37) and hairpin loop (16/37)-based on the core adenosine. Considering the systematic comparative analysis performed in this study, future thorough biochemical research is warranted to determine the role of m6A modification during the replication and infection of ALV-J. These conservation and distribution analysis of the DRACH motif for potential m6A sites in ALV-J would provide a foundation for the future intervention of ALV-J infection and m6A modification.

The Development of a Multienzyme Isothermal Rapid Amplification Assay to Visually Detect Duck Hepatitis B Virus.

Duck hepatitis B virus (DHBV) is widely prevalent in global ducks and has been identified in Chinese geese with a high prevalence; the available detection techniques are time-consuming and require sophisticated equipment. In this study, an assay combining multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) was developed for the efficient and rapid detection of DHBV. The primary reaction condition of the MIRA assay for DHBV detection was 10 min at 38 °C without a temperature cycler. Combined with the LFD assay, the complete procedure of the newly developed MIRA assay for DHBV detection required only 15 min, which is about one-fourth of the reaction time for routine polymerase chain reaction assay. And electrophoresis and gel imaging equipment were not required for detection and to read the results. Furthermore, the detection limit of MIRA was 45.6 copies per reaction, which is approximately 10 times lower than that of a routine polymerase chain reaction assay. The primer set and probe had much simpler designs than loop-mediated isothermal amplification, and they were only specific to DHBV, with no cross-reactivity with duck hepatitis A virus subtype 1 and duck hepatitis A virus subtype 3, goose parvovirus, duck enteritis virus, duck circovirus, or Riemerella anatipestifer. In this study, we offer a simple, fast, and accurate assay method to identify DHBV in clinical serum samples of ducks and geese, which would be suitable for widespread application in field clinics.

First report of goose circovirus identified in ducks from China.

Goose circovirus (GoCV) is a common pathogen that causes immunosuppression and promotes secondary infections with other infectious agents in geese worldwide. In the present study, we identified GoCV in 2 out of 93 duck flocks from China and successfully sequenced the complete genomes of 2 strains (AH22du and HN20du). The whole genome of the two strains shared a high identity of 90.5 to 98.63% with China GoCV reference, and low identity of 58.98% with DuCV reference, respectively. Phylogenetic tree constructed on the two and other genome sequences of GoCV revealed three main branches. Both strains sequenced in this study were distributed on different sub-branches with most other Chinese GoCV strains, and AH22du clustered into an independent sub-branch within the cluster. Recombination analysis predicted that HN20du might potentially recombine from the major parent of yk4 (Zhejiang Province, China, 2007) and minor parent of GD/YJ/g2 (Guangdong Province, China, 2020). To the best of our knowledge, this is the first report of GoCV in ducks from China. This broadened host spectrum of GoCVs requires attention from the waterfowl industry and researchers.

Genetic and recombination heterogeneity of Canine Bufaviruses detected in diarrheal dogs in China.

Bufavirus (BuV) was first identified in feces from children with acute diarrhea, and a genetically related Canine bufavirus (CBuV) was first reported in Italy in 2018. In this study, through the investigation of CBuV in 622 anal swabs from dogs with diarrhea symptoms collected from various provinces in northern, central and eastern China during 2018-2022, 14 samples were detected to be positive. And 5 samples were from dogs co-infected with other canine diarrhea related viruses, which consist of CPV-2, CDV and CCoV. The complete genome sequences (4219 nt) of the fourteen strains were amplified and sequenced. Through comparative analysis with 51 reference BuV strains, six strains might recombinate from the CBuV strains (HUN/2012/22, CaBuV/9AS/2005/ITA and CaBuV/35/2016/ITA) in Hungary and Italy as the parents, and two genetic recombination events from various parents were predicted to occur on the BUV-422 strain. Combined analyzing the phylogenetic tree and sequence alignment, it was found that these CBuVs are highly conserved in the nonstructural protein NS1, but indeed various amino acid mutation sites in the capsid protein VP2, and even some amino acid sites coincide with putative protein plastic regions and potential epitopes. The BUV-422 and BUV-512 strains show sequential mutation sites identical to the divergent strains of CaBuV/9AS/2005/ITA and CaBuV/35/2016/ITA. This study would enrich the molecular data of CBuV in China and provide essential reference for the epidemiological research and vaccine development of CBuV in the future.

Molecular analysis of Gyrovirus galga1 variants identified from the sera of dogs and cats in China.

Gyrovirus galga1 (GyVg1), a member of the Anelloviridae family and Gyrovirus genus, has been detected in chicken and human tissue samples. In this study, the DNA of GyVg1-related gyroviruses in the sera of six dogs and three cats from Central and Eastern China was identified using PCR. Alignment analysis between the nine obtained and reference GyVg1 strains revealed that the genome identity ranged from 99.20% (DOG03 and DOG04 strains) to 96.17% (DOG01 and DOG06 strains). Six recombination events were predicted in multiple strains, including DOG01, DOG05, DOG06, CAT01, CAT02, and CAT03. The predicted major and minor parents of DOG05 came from Brazil. The DOG06 strain is potentially recombined from strains originating from humans and cats, whereas DOG01 is potentially recombined from G17 (ferret-originated) and Ave3 (chicken-originated), indicating that transmissions across species and regions may occur. Sixteen representative amino acid mutation sites were identified: nine in VP1 (12 R/H, 114S/N, 123I/M, 167 L/P, 231 P/S, 237 P/L, 243 R/W, 335 T/A, and 444S/N), four in VP2 (81 A/P, 103 R/H, 223 R/G, and 228 A/T), and three in VP3 (38 M/I, 61 A/T, and 65 V/A). These mutations were only harbored in strains identified in dogs and cats in this study. Whether this is related to host tropism needs further investigation. In this study, GyVg1 was identified in the sera of dogs and cats, and the molecular characteristics prompted the attention of public health.

Complete genome sequence of a variant porcine epidemic diarrhea virus strain isolated in China.

Since October 2010, an outbreak of porcine epidemic diarrhea (PED) has been observed in some provinces of China. Here we report the complete genome sequence of porcine epidemic diarrhea virus (PEDV) strain LC, which was recently isolated from sucking piglets that suffered from severe watery diarrhea in Guangdong. It will help in understanding the epidemiological and molecular characteristics of PEDV in China.

Complete genome sequence of porcine circovirus 2d strain GDYX.

Porcine circovirus type 2 (PCV2) is the etiologic agent of porcine circovirus-associated disease, and it is mainly divided into five genotypes. Here, we report the complete genome sequence of PCV2 strain GDYX, which belongs to PCV2d and has a unique amino acid variation at position 169 (S to G).

Complete genome sequence of an avian leukosis virus isolate associated with hemangioma and myeloid leukosis in egg-type and meat-type chickens.

Subgroup J avian leukosis virus (ALV-J) was first isolated from meat-type chickens that developed myeloid leukosis (ML). In recent years, field cases of hemangioma (HE) or HE and ML, rather than ML alone, have been reported in commercial layer flocks exposed to ALV-J with a high incidence in China. Here we report the complete genomic sequence of an ALV-J isolate that caused both HE and ML in egg-type and meat-type chickens in China. These findings will provide additional insights into the molecular characteristics in genomes, host range, and pathogenicity of ALV-J.

Complete genome sequence of porcine reproductive and respiratory syndrome virus strain QY2010 reveals a novel subgroup emerging in China.

QY2010 is a highly pathogenic North American-type porcine reproductive and respiratory syndrome virus (PRRSV). The complete genome sequence shows that QY2010 shares low sequence identity (60 to 88.7%) to all known PRRSV isolates. Phylogenetic analyses further reveal that QY2010 constitutes a novel subgroup within the North American genotype of PRRSV.

Complete genome sequence of a recombinant nephropathogenic infectious bronchitis virus strain in China.

Recently, nephropathogenic infectious bronchitis virus (IBV) outbreaks have occurred in commercial broiler flocks and have been associated with a high incidence and morbidity in China. The CK/CH/Zhejiang/06/10 strain (IBV-YX10) was isolated from a 12-day-old broiler chicken in a flock of chickens with swollen speckled kidneys and distended ureters filled with uric acid in China in 2010. Here we reported the complete genomic sequence of the IBV-YX10 which was a natural recombinant nephropathogenic infectious bronchitis virus strain. These findings will contribute additional insights into the molecular characteristics of evolving IBV genomes and the need for effective control of IBV in China.

Complete genome sequence of a porcine orthoreovirus from southern China.

Porcine orthoreoviruses belong to the family Reoviridae and cause mainly mild enteritis in piglets. We present here the complete genome sequence of a novel porcine orthoreovirus strain (GD-1) isolated from a piglet in southern China. Our data will facilitate future investigations of the molecular characteristics and epidemiology of porcine orthoreoviruses.

Complete genome sequence of a Newcastle disease virus strain isolated from broiler breeder flocks in China.

In 2010 and 2011, several devastating Newcastle disease (ND) outbreaks occurred in China, affecting broilers, layers, and breeders. The CK-JSX1-201005 virus was isolated from broiler breeder flocks vaccinated with the classical ND virus (NDV) vaccine program, but laying rate decreased from 80% to 30 to 40% in the clinic. Here, we report the complete genome sequence and molecular characteristic of the CK-JSX1-201005 NDV. These findings provide additional insights into the genetic variation of NDV circulating in China and are useful for vaccine development for NDV.

Complete genome sequence of two variant porcine reproductive and respiratory syndrome viruses isolated from vaccinated piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to affect the Chinese swine industry. Since 2006, variant PRRSV strains sharing two unique discontinuous deletions of 30 amino acids in the nonstructural protein Nsp2 have become dominant in Chinese swine herds and have caused huge economic losses to the swine industry in China. Here we report the complete genome sequence of two novel PRRSV variants isolated from vaccinated piglets with additional amino acid deletions in Nsp2.

Complete genome sequence of a J subgroup avian leukosis virus isolated from local commercial broilers.

Subgroup J avian leukosis virus (ALV-J) isolate GDKP1202 was isolated from a 50-day-old local yellow commercial broiler in the Guangdong province of China in 2012. Here we report the complete genomic sequence of the GDKP1202 isolate, which caused high mortality, serious growth suppression, thymic atrophy, and liver enlargement in commercial broilers. A novel potential binding site (5'-GGCACCTCC-3') for c-myb was identified in the GDKP1202 genome. These findings will provide additional insights into the molecular characteristics in the genomes and pathogenicity of ALV-J.

Complete genome sequence analysis of a natural reassortant infectious bursal disease virus in China.

A novel isolate of infectious bursal disease virus (IBDV) was designated GX-NN-L. The GX-NN-L IBDV was a very virulent infectious bursal disease virus (vvIBDV) isolated from broiler flocks in Guangxi province, China, in 2011. The GX-NN-L IBDV caused high mortality, immunosuppression, low weight gain, and bursal atrophy in commercial broilers. Here, we report the complete genome sequence of the GX-NN-L IBDV, a reassortment strain with segments A and B derived from very virulent strains and attenuated IBDV, respectively. These findings from this study provide additional insights into the genetic exchange between attenuated and very virulent strains of IBDV and continuous monitoring of the spread of the virus in chicken.