A review of the influence of environmental pollutants (microplastics, pesticides, antibiotics, air pollutants, viruses, bacteria) on animal viruses.

Microorganisms, especially viruses, cause disease in both humans and animals. Environmental chemical pollutants including microplastics, pesticides, antibiotics sand air pollutants arisen from human activities affect both animal and human health. This review assesses the impact of chemical and biological contaminants (virus and bacteria) on viruses including its life cycle, survival, mutations, loads and titers, shedding, transmission, infection, re-assortment, interference, abundance, viral transfer between cells, and the susceptibility of the host to viruses. It summarizes the sources of environmental contaminants, interactions between contaminants and viruses, and methods used to mitigate such interactions. Overall, this review provides a perspective of environmentally co-occurring contaminants on animal viruses that would be useful for future research on virus-animal-human-ecosystem harmony studies to safeguard human and animal health.

A new rapid and sensitive method for detecting chicken infectious anemia virus.

Since the chicken infectious anemia virus (CIAV) was discovered in 1979, which has been reported as an economically significant and immunosuppressive poultry disease in the world. A novel clinical detection method for the prevention and control of CIAV in the poultry sector is urgently needed. Here, we established a real-time recombinase-aided amplification assay (RAA) for CIAV on-site with a rapid, highly sensitive, strongly specific, low-cost, and simple operational molecular diagnosis detection method. The primers and probe were developed using the CIAV VP2 gene sequence, which has a 117-bp specific band. This assay, which could be carried out at 41°C and completed in 30 min without cross-reactivity with other viruses, had the lowest detection limit of 10 copies of CIAV DNA molecules per reaction. Furthermore, the kappa value of this assay was 0.947, the sensitivity was 93.33%, and the specificity was 100% when compared to the real-time quantitative polymerase chain reaction assay (real-time qPCR). These results indicate that using a real-time RAA assay to detect CIAV on-site could be beneficial. In the future, the real-time RAA test may be a regular assay for the prevention and control of CIAV, as well as help the reduction of economic losses in the poultry business.

Toosendanin suppresses African swine fever virus replication through upregulating interferon regulatory factor 1 in porcine alveolar macrophage cultures.

African swine fever virus (ASFV) is a highly infectious and lethal swine pathogen that causes severe socio-economic consequences in affected countries. Unfortunately, effective vaccine for combating ASF is unavailable so far, and the prevention and control strategies for ASFV are still very limited. Toosendanin (TSN), a triterpenoid saponin extracted from the medicinal herb Melia toosendan Sieb. Et Zucc, has been demonstrated to possess analgesic, anti-inflammatory, anti-botulism and anti-microbial activities, and was used clinically as an anthelmintic, while the antiviral effect of TSN on ASFV has not been reported. In this study, we revealed that TSN exhibited a potent inhibitory effect on ASFV GZ201801-38 strain in porcine alveolar macrophages (PAMs; EC50 = 0.085 µM, SI = 365) in a dose-dependent manner. TSN showed robust antiviral activity in different doses of ASFV infection and reduced the transcription and translation levels of ASFV p30 protein, viral genomic DNA quantity as well as viral titer at 24 and 48 h post-infection. In addition, TSN did not affect virion attachment and release but intervened in its internalization in PAMs. Further investigations disclosed that TSN played its antiviral role by upregulating the host IFN-stimulated gene (ISG) IRF1 rather than by directly inactivating the virus particles. Overall, our results suggest that TSN is an effective antiviral agent against ASFV replication in vitro and may have the potential for clinical use.

Development and Application of a Reverse-Transcription Recombinase-Aided Amplification Assay for Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus currently widespread worldwide in the swine industry. Since PEDV was discovered in China in 1984, it has caused huge economic losses in the swine industry. PEDV can infect pigs of all ages, but piglets have the highest infection with a death rate as high as 100%, and the clinical symptoms are watery diarrhea, vomiting, and dehydration. At present, there is not any report on PEDV detection by RT-RAA. In this study, we developed an isothermal amplification technology by using reverse-transcription recombinase-aided amplification assay (RT-RAA) combined with portable instruments to achieve a molecular diagnosis of PEDV in clinical samples from China. By designing a pair of RT-RAA primers and probes based on the PEDV N gene, this method breaks the limitations of existing detection methods. The assay time was within 30 min at 41 °C and can detect as few as 10 copies of PEDV DNA molecules per reaction. Sixty-two clinical tissue samples were detected by RT-qPCR and RT-RAA. The positive and negative rates for the two methods were 24.19% and 75.81%, respectively. Specificity assay showed that the RT-RAA had specifically detected PEDV and was not reactive for porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), swine flu virus (SIV), or porcine Japanese encephalitis virus (JEV). The results suggested that RT-RAA had a strong specificity and high detection sensitivity when combined with a portable instrument to complete the detection under a constant temperature of 30 min, which are more suitable for preventing and controlling PEDV onsite in China.

Prevention and Control Strategies of African Swine Fever and Progress on Pig Farm Repopulation in China.

African swine fever (ASF) is a devastating disease in domestic and wild pigs. Since the first outbreak of ASF in August 2018 in China, the disease has spread throughout the country with an unprecedented speed, causing heavy losses to the pig and related industries. As a result, strategies for managing the disease are urgently needed. This paper summarizes the important aspects of three key elements about African swine fever virus (ASFV) transmission, including the sources of infection, transmission routes, and susceptible animals. It overviews the relevant prevention and control strategies, focusing on the research progress of ASFV vaccines, anti-ASFV drugs, ASFV-resistant pigs, efficient disinfection, and pig farm biosecurity. We then reviewed the key technical points concerning pig farm repopulation, which is critical to the pork industry. We hope to not only provide a theoretical basis but also practical strategies for effective dealing with the ASF epidemic and restoration of pig production.

Occurrence and molecular identification of Giardia duodenalis from stray cats in Guangzhou, southern China.

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), ß-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.

Assessing the efficacy of an inactivated chicken anemia virus vaccine.

BACKGROUND: Chicken anemia virus (CAV) is an immunosuppressive virus that causes chicken infectious anemia (CIA) which is a highly contagious avian disease. CAV causes major economic losses in the poultry industry worldwide. The current CAV vaccine is a live attenuated strain administered in the drinking water that risks horizontal infection of other chickens. The purpose of this study was to develop a novel vaccine against CAV that can be administered safely using a highly pathogenic isolate inactivated with ß-propiolactone hydrolysis that would protect chicks from CAV. METHODS: Hens were vaccinated twice intramuscularly with a novel CAV GD-G-12 inactivated vaccine and the humoral immune responses of the hens and offspring were monitored by ELISA. A heterologous intramuscular challenge using the CAV strain GD-E-12 was conducted in the chicks hatched from vaccinated or unvaccinated hens. RESULTS: The vaccine strain, GD-G-12, was shown to be highly pathogenic prior to inactivation evidenced by thymic atrophy and bleeding, and weight loss. The inactivated vaccine was considered safe and showed no signs of pathogenicity. High titers of CAV specific antibodies were detected in the vaccinated hens and in their chicks, indicating vertical transfer of maternal antibodies. Furthermore, the chicks hatched from vaccinated hens were resistant to a heterologous CAV challenge and showed no signs of weight loss and thymic atrophy and bleeding. CONCLUSION: Our studies are proof of principle that inactivated GD-G-12 might be a novel vaccine candidate to prevent CAV infection, and highlight the utility of using an inactivated virus for this vaccine.

Genotyping of Giardia duodenalis isolates from dogs in Guangdong, China based on multi-locus sequence.

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), ß-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.

The zoonotic risk of Ancylostoma ceylanicum isolated from stray dogs and cats in Guangzhou, South China.

Canine and feline hookworm infection is endemic in many countries with zoonotic transmission representing a potentially significant public health concern. However, there is limited data available on the zoonotic transmission of canine and feline hookworms in China. This study was conducted to evaluate the zoonotic risk of Ancylostoma ceylanicum isolated from stray dogs and cats in Guangzhou, south China. Primer pairs CAF/CAR were designed to amplify complete ITS sequences of obtained A. ceylanicum. The results were compared with fourteen ITS reference sequences of human-derived A. ceylanicum registered in GenBank, and phylogenetic trees were established by using NJ and ML methods. The sequence similarity of three dog-derived and five cat-derived A. ceylanicum with fourteen human-derived A. ceylanicum were 96.8%~100% and 97.8%~100%, respectively. Phylogenetic analysis placed A. ceylanicum isolated from dogs and cats in the same group with A. ceylanicum human isolates. Due to the ability of A. ceylanicum to cause a patent infection in humans, the zoonotic risk arising from dog and cat reservoirs to communities in this region should be determined.

Identification of a chicken anemia virus variant-related gyrovirus in stray cats in china, 2012.

The chicken anemia virus (CAV), is a known member of the genus Gyrovirus and was first isolated from chickens in Japan in 1979. Some reports have also demonstrated that CAV can be identified in human stool specimens. In this study, a variant of CAV was detected using PCR with CAV-based primers in fecal samples of stray cats. The genome of CAV variant was sequenced and the results suggest that it could be a recombinant viral strain from parental CAV strains JQ690762 and AF311900. Recombination is an important evolutionary mechanism that contributes to genetic diversification. These findings indicate that CAV variant might have originated from CAV-infected chickens. The epidemiology and pathogenesis of this novel virus remains to be elucidated. This study underscores the importance of CAV surveillance and it presents the first evidence suggesting the possibility of CAV homologous recombination in cat.

Phylogenetic and molecular characterization of chicken anemia virus in southern China from 2011 to 2012.

Chicken anemia virus (CAV) is an important pathogen that causes severe immunosuppression in young chickens. We have characterized 13 CAVs isolated from different commercial farms in southern China between 2011 and 2012. We discovered 92 variable residues compared to 37 other CAV complete genome sequences from other parts of the world listed in GenBank; these residues have not been previously observed. All of the Chinese CAV genomes that were characterized in this study had a glutamine at position 394, a hallmark of highly pathogenic CAVs. We also discovered that intra-group genetic recombination plays a role in generating genetic diversity in natural populations of CAV. The GD-J-12 isolate was a possible recombinant between GD-C-12 and GD-M-12 in the genomic region that encompassed both the coding and non-coding regions.

Molecular identification of Ancylostoma caninum isolated from cats in southern China based on complete ITS sequence.

Ancylostoma caninum is a blood-feeding parasitic intestinal nematode which infects dogs, cats, and other mammals throughout the world. A highly sensitive and species-specific PCR-RFLP technique was utilised to detect the prevalence of A. caninum in cats in Guangzhou, southern China. Of the 102 fecal samples examined, the prevalence of A. caninum in cats was 95.1% and 83.3% using PCR-RFLP and microscopy, respectively. Among them, the prevalence of single hookworm infection with A. caninum was 54.90%, while mixed infections with both A. caninum and A. ceylanicum were 40.20%. Comparative analysis of three complete ITS sequences obtained from cat-derived A. caninum showed the same length (738 bp) as that of dog-derived A. caninum. However, the sequence variation range was 98.6%-100%, where only one cat isolate (M63) showed 100% sequence similarity in comparison with two dog-derived A. caninum isolates (AM850106, EU159416) in the same studied area. The phylogenetic tree revealed A. caninum derived from both cats and dogs in single cluster. Results suggest that cats could be the main host of A. caninum in China, which may cause cross-infection between dogs and cats in the same area.

Application of HRM assays with EvaGreen dye for genotyping Giardia duodenalis zoonotic assemblages.

A high-resolution melting (HRM) assay was applied to distinguish between Giardia duodenalis assemblages A and B from human and dog feces based on the triosephosphate isomerase gene (tpi). The genomic DNAs were selected from assemblages A (WB) and B (GS) as reference and plasmids were constructed. The reference plasmids and genomic DNAs from 15 Giardia-positive samples were analyzed by HRM assay. This was followed by separate real-time PCR assays specific for assemblages A and B using EvaGreen (EG) to identify PCR products by melting-point analysis. Our results indicate that PCR with HRM in a one-step closed-tube method is a reliable diagnostic method for G. duodenalis zoonotic assemblage identification and more rapid than restriction length polymorphism analysis and direct sequence analysis, HRM is specific, sensitive, reproducible, and rapid. This study is the first use of EG dye for Giardia genotyping. This assay is a promising approach to determine the presence and genotype of Giardia based on a highly variable gene.

Genotype identification and prevalence of Giardia duodenalis in pet dogs of Guangzhou, Southern China.

Giardia duodenalis is a flagellated parasite and is considered one of the most common causes of protozoal diarrhea in both humans and animals worldwide. This paper represents the first study of the prevalence of G. duodenalis in pet dogs in Guangzhou, China. Faecal samples (209 specimens) were obtained from young (<6 months old), adult (6 months to 3 years) and elder dogs (>3 years old). 8.61% (18/209) faecal samples were recorded positive using microscopy examination, and 11.00% (23/209) using PCR. The prevalence was significantly higher in diarrheic dogs (26.31%) compared with non-diarrheic dogs (5.10%), while it was higher in young (25.58%) than both adult (7.37%) and elder (7.04%) dogs and the difference was statistically significant (P<0.05). The prevalence in male dogs 11.30% (13/115) was higher than females 10.87% (10/92), and in suburban dogs (12.15%) higher than urban 9.80%, but the difference was not statistically significant (P>0.05). Sequence analysis of the 23 PCR-positive samples revealed the presence of Assemblage D (18/23), and zoonotic Assemblage A (5/23). The present investigation reported a high infection rate of G. duodenalis in pet dogs, especially in young dogs. Genotypic characterization demonstrated that the zoonotic Assemblage A was found, a fact that poses a potential risk of G. duodenalis transmission from pet dogs to humans. It is suggested that pet owners should take appropriate hygiene measures to prevent and control giardiasis in this region.