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To develop a multiplex fluorescent quantitative RT-PCR for the detection of porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and swine acute diarrhea syndrome coronavirus (SADS-CoV), in this study, specific primers/probes were designed based on the conserved regions of M, M and N gene sequences of PEDV, PDCoV and SADS-CoV, respectively. After optimization of the reaction conditions, a multiplex fluorescent quantitative RT-PCR for PEDV, PDCoV and SADS-CoV was established. The results of specificity assay showed that the method was positive for detection of PEDV, PDCoV and SADS-CoV, and negative for detection of porcine transmissible gastroenteritis virus, porcine rotavirus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine parvovirus, classical swine fever virus and foot-and-mouth disease virus. The results of sensitivity assay showed that the detection limit of this method for PEDV, PDCoV, and SADS-CoV plasmids standard was 1.0x101 copies/L, and had a good linear relationship with their Ct values in the range of 101 copies/L to 106 copies/L. The results of repeatability assay showed that the coefficients of variation (CVs) of intra- and inter-assay reproducibility ranged from 0.33% to 2.53%, indicating good repeatability and stability. To evaluate the effects of the developed method, 100 clinical samples collected from different parts of Henan province were used for detection of these three viruses and compared with those of single RT-PCR and standard methods. The results of multiplex fluorescent quantitative RT-PCR showed that the positive rates of PEDV, PDCoV and SADS-CoV were 38% (38/100), 14% (14/100) and 5% (5/100), respectively. There was no mixed infection. The coincidence rate with the standard detection methods of PEDV and PDCoV was 100%, and the sensitivity was higher than that of single RT-PCR. In this study, a specific, sensitive and rapid multiplex fluorescent quantitative RTPCR method was established for the first time, which could be used for the differential detection of PEDV, PDCoV and SADS-CoV, and laid a foundation for the differential diagnosis and control of porcine diarrheal diseases.
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Objective: Epidemiology and genetic variations of the infectious bronchitis virus(IBV) in Fujian province were studied. Method: Two strains of virus isolated from the diseased chickens in Fujian in 2021 were identified by chicken embryo pathogenicity test, electron microscope observation, and RT-PCR. S1 genes of the isolates were cloned, sequenced, and analyzed using biological software. Result: The two IBV strains were code named FJ-NP01 and FJ-FZ01. The full length of S1 of FJ-NP01 was 1 629 nt encoding 543 amino acids, and that of FJ-FZ01, 1 620 nt encoding 540 amino acids. The S1 gene cleavage site of FJ-FZ01 was HRRRR, same as all reference strains of genotype I branch;while that of FJ-NP01 HRRKR differed from the reported site of IBV isolated from genotype IV but same as that of TC07-2 reference strain of genotype VI. The homology of nucleotide and amino acid between the two isolates was 83.2% and 79.6%, respectively, but merely 75.7%-76.3%and 77.1%-83.5% with the Mass-type conventional vaccines H120 and H52, respectively. Further analysis showed that FJ-NP01was from a recombination event between CK CH GD LZ12-4 and L-1148, the homology of nucleotide acid between 1438-1506 nt of FJ-NP01 with CK CH GD LZ12-4 was 97%, and 95.9% between the other nucleotide acid of S1 gene with L-1148. Conclusion: It appeared that the IBV epidemic experienced in the province was complex in nature and that the existing Mass vaccines would not provide sufficient immune protection to deter the spread.
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Purpose: To investigate the epidemic variation of porcine epidemic diarrhea virus (PEDV) strains in Sichuan Province, and to analyze the causes of poor vaccination effect. Methods: Piglet intestinal samples were collected from a pig farm in Sichuan Province for PCR detection, virus purification, determination of virus titer and virus infection experiments. Whole genome sequencing of isolated strains was determined. The S gene sequence of the isolated strain was compared with the strains from other regions and vaccine strains, and the phylogenetic tree was established. The amino acid site variation of S protein between the isolated strain and the classical vaccine strain CV777 was compared. Results: A PEDV strain was successfully isolated and named as PEDV SNJ-P. The determination of virus titer was 1..107.5/100 L. Animal infection experiments showed that the isolated strain could cause diarrhea, dehydration and other symptoms and lead to death in piglets. Genome sequencing and phylogenetic tree analysis showed that the whole gene of PEDV SNJ-P strain was 28003 bp, and the genotype of the strain was S non-INDEL type. The strains were closely related to the strains of PEDV-WS, CH/JLDH/2016 and CH/HNLH/2015 isolated from China, and were relatively distant with the same type vaccine strain, and were far from the classical vaccine strain. Compared with the classical vaccine strain CV777, the S protein of SNJ-P strain had multiple amino acid mutations, deletions and insertions. Conclusion: Due to the continuous variation of strains, SNJ-P strain is far from the vaccine strain, and the current vaccines cannot provide effective protection. The results of this study are expected to provide reference for the study of PEDV strains and vaccine development in China.
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Horses may act as incidental host and experience silent infection following spillover from humans with COVID-19. SARS-CoV-2-infected humans should avoid close contact with equids during the time of their illness.
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Mucormycosis is a rare type of fungal infection commonly known as zygomycosis, the infection tends to crop up more commonly in individuals with low and weakened immunity level, if left untreated, the mucormycosis can be life-threatening and fatal. Mucormycosis previously known as zygomycosis is a consequential type of infection caused by several mildews known as micromycetes. The revised taxonomical studies revealed that the micromycetes causing the infections are classified as the species of phylum Glomeromycota, class Glomeromycetes, subphylum Mucoromycotina, order Mucorales. The genera of Rhizopus, Mucor, Lichtheimia, Cunninghamella, Rhizomucor, and Apophysomyces, constitute the causative agents of the majority of cases of mucormycosis. The angioinvasive type of disorder caused by mucormycosis is further classified as Mucorales. The patients with Diabetes ketoacidosis and diabetes mellitus are at high-risk factors, followed by the patients with organ transplant, immunocompromised disease, and malignancy. The route of exposure to Mucormycosis may be through the wounded infection that can be pneumonic, or dermal in origin. In the ectodermal form, the fungal organism can invade the skin through open or puncture wounds, or the laceration on the skin. However, the infection has a high mortality rate, the key to successful treatment is early diagnosis, and administration of antifungal drugs, with extensive therapy, followed by surgical debridement of the infection. The morbidity and mortality rate are still at a high number, due to the negligence of the patient to seek medical treatment. Hence the early diagnosis and treatment with antifungal drugs with surgical debridement is a must. The efficacy of oral and venous formulations in the treatment of mucorales is still under debate. Despite the aggressive therapy, the mortality rate is increasing worldwide. The studies have to be conducted to invent the fastest treatment protocol for the treatment of Mucormycosis.
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Objective: The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. Method: The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value. The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. Result: Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1..102 copies/L. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. Conclusion: In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.
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In order to understand the genomic characteristics and molecular genetic diversity of porcine epidemic diarrhea virus(PEDV) in Guangxi in recent years, 11 pairs of specific primers were designed to detect the whole genome of PEDV GXNN isolated from porcine diarrhea in Nanning, Guangxi, China, and similarity comparison, genetic evolution, gene variation and S gene recombination were also analyzed. The results showed that full length of the GXNN strain was 28 035 bp, had similar genomic characteristics with other PEDV isolates, about 96.4%-98.7% nucleotide similarity with different reference strains, and the nucleotide similarity of S, ORF3, M and N genes was 93.7%-98.9%, 90.9%-99.4%, 97.4%-99.7% and 95.6%-99.2%;the amino acid similarity of them was 92.9%-99.5%, 91.3%-99.1%, 97.4%-99.1% and 96.4%-99.5%. GXNN is closely related to most domestic isolates in recent years. Phylogenetic tree showed that GXNN closely related to most strains isolated in China recent years, belonged to GII-b subtype. However, it was low relatedness to classic vaccine strains, domestic early epidemic strains, foreign epidemic strains and Guangxi CH-GX-2015-750 A, they belong to different subtypes. Compared with the 5 vaccine strains, the S gene of GXNN stain has a large variation, by inserting amino acid Q at positions 118 844 and 905 sizes, four unique amino acid mutations in the core neutralizing epitope(COE)region and the main epitope region, and 14 mutations in other regions. 126 T/A, 199 A/V and 103 T/A site mutations of ORF3, M and N genes were happened at position 126, 4 D4 region and PN-D4 region, respectively. Recombination analysis revealed that there was a potential recombination region in the hypervariable region of S gene at 826-3 142 nt. This study successfully obtained the complete genome sequence of a PEDV strain, and analyzed its genetic variation and provided a reference for PEDV molecular epidemiology research and new vaccine development.
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Objective: To learn the prevalence and genotypic diversity of canine coronavirus(CCoV)in healthy and diarrhea dogs in Shandong Province.
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In order to build a specific, sensitive and rapid detection method for PAstV3 detection, the PAstVB gene sequences in Genbank were used and the conserved region in ORFlb was selected to design specific primers and TaqMan probe. Clinical stool samples were collected and preliminary detected by this newly established real-time RT-PCR method after reaction systems and conditions optimization. This detection method established in this study has a good linear relationship with the standard curve, with R2 value up to 0.9971. The sensitivity is 100 times higher than conventional PCR method, The variation co-efficient of in-batch and inter-batch repeatability test is less than 2.0%, indicating good repeatability. The detection results of Clinical samples showed that the positive rate of this method is higher than conventional PCR method. The establishment of this method provides a rapid detection means for PAstV3 laboratory diagnosis and epidemiological investigation.
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Avian infectious bronchitis (IB) is one of the acute and highly contagious upper respiratory tract infectious diseases in poultry caused by the Infectious bronchitis virus (IBV), which significantly affects the health and development of world poultry farming industry. IBV RNA polymerase lacks a complete correctional function and is prone to gene mutation and RNA-RNA recombination during the replication process, resulting in the emergence of new serotypes, genotypes and mutant strains. The continuous generation of recombinant strains through homologous recombination between strains also complicates the prevention and control of IB. Therefore, monitoring the genetic evolutionary characteristics of circulating strains and evaluating the protective effect of commonly used vaccines against local circulating strains of IBV are the keys to preventing and controlling this disease.
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This special issue consists of 17 papers dealing with issues animal health (captive and wild primates), environmental health (rain forests and mountain areas), and human health (the role of religion in One Health, lessons from the Hanuman langur (Semnopithecus entellus) and other human-non-human primate interactions,and Covid-19).
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This paper presents a review of most important zoonotic diseases that are threatening human World population in the first 20 years of XXI century. Zoonoses diseases naturally transmitted through several modes from vertebrate animal hosts to humans. SARS-CoV-Z - severe acute respiratory syndrome coronavirus 2, was identified as the cause of an outbreak of COVID-2 pandemic in humans in 2019/2020. Coronavirus positive Chinese bats and an unrecognized yet natural reservoir of emerging SARS-Z, are indicated as a primary source of infection. So far, there is no evidence that companion or farm animals can become infected by contact with a sick/infected person, so SARS-2 virus strains isolated from humans are not zoonotic. This review contains a description of SARS-2 virus structure, genetic diversity, structure and function of viral proteins, including class I viral fusion protein S. The review also includes an assessment of epidemiology of SARS-2 infection, criteria and epidemiological interactions, perspectives on emerging zoonoti'c disease research in contact with public health service. More closed cooperation between different services, including Veterinary Services, with WHO and OIE international standards, as eg. One Health partnership, is essential to avoid or minimize risk of new infections in future.
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To understand the genetic diversity of porcine deltacoronavirus(PDCo V) in Guangxi Province, clinical diarrhea samples were collected from suspected piglets in Guangxi Province from2017 to 2019, detected by RT-PCR for PDCoV, and the positive samples were used for amplification and sequence of S, M, N genes. Finally, 16 S, M and N gene sequences of PDCoV were obtained. Homology analysis showed that the S, M, N gene nucleotide identity among Guangxi strains were 95.8% -99.9%, 95.9%-100% and 97.9%-99.9%, respectively. The nucleotide identity of S, M and N genes among Guangxi strains and other reference strains were 95.1%-100%, 95.0%-100%and 96.3%-99.9%, respectively. Sequence alignment showed that S1 protein existed amino acid mutations and insertions, and there were some variations among different epidemic strains. Phylogenetic trees based on S, M and N genes obtained similar topological diagram and all strains could be divided into Group I, Group II and GroupIII, of which Group I came from USA, Japan and Korea, Group II came from China, and Group III came from China, Vietnam, Laos and Thailand. Most strains from Guangxi Province distributed in Group II, individual strain distributed in Group III and some strains formed a single small branch. The evolutionary rates of S, M and N genes of Guangxi strains and other reference strains were 2.57 x 10-4, 2.07 x 10-4, 1.70 x 10-4 substitutions/site/year, respectively, showing that the evolutionary rate of S gene was the fastest. The results indicated that the S, M, N genes of PDCo V strains from Guangxi Province had some variations and existed genetic diversity.
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Emergence of avian infectious bronchitis virus (IBV) variants with altered tissue tropism and host range has been reported from different parts of the world. Little is known about the different IBV variants existing and emerging in India. To explore the same, an IBV isolate, namely B17 isolated from backyard chicken in Tamil Nadu was used in the present study. The complete genome of B17 was sequenced and its phylogenetic relationship with the existing vaccine strain genotypes was analysed. The phylogenetic analysis of both S1 gene and complete genome sequence grouped B17 under Mass41 genotype comprising of M41, Beaudette, H120 and H120 variant with bootstrap value of 95-100%. Further, genomic analysis of B17 revealed the possibilities of emergence of the same from H120 vaccine strain through mutations at various genes.
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Revitalization of local health traditions (RLHT) has become an inevitable aspect of human wellbeing in the post COVID era. An ethnobotanical survey was carried out to collect information on local health traditions and cultural reflections associate with the age old use of wild edible fruits (WEFs) from common plants in Melur Region of Madurai district, TamilNadu, India as the habit of consuming WEFs is quite common among people in this region and has not been completely abandoned in particular among the age old people. Information presented in this paper has been gathered from local people using an integrated approach of botanical collections, group discussions and interviews with questionnaires during the period from Apr 2021 to Mar 2022. As much as 29 informants were interviewed, among the informants 6 were local health-care practitioners (Vidiyars). Studies on the use of WEFs from common plants in Melur resulted in collection and documentation of information on a total of 34 ethnomedicinal plant species distributed across 20 families. Medicinal plants used by local people are listed with scientific name, family, local name, plant part(s) used, mode of consumption and preparation and medicinal uses. Data collected during the study clearly indicates that fresh parts of the plant (Fruit (Ripe/ Unripe)) were more preferred in general for the preparation of medicinal formulations by the local health practitioners. Documented ethnomedicinal plants were mostly used to cure long term complications associated with diabetics, gastrointestinal disorders, skin diseases, poison bites and nervous disorders. Howsoever, results of this study is clear record to the claim that the local people still depend on medicinal plants to overcome situations like COVID pandemic as fruits from most of the plants documented serve as natural source of immune boosters. Further, in-depth studies (both In-silico and Pre Clinical trials) are expected to bring to limelight the hidden quantum of bioactive compounds in the fruits these medicinal plants and their therapeutic potential.
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The normal management of plant germplasm conservation laboratories involves carrying out numerous and diverse activities, which were affected by the coronavirus pandemic (COVID-19). The objective of this publication was to review the evolution of the cassava in vitro germplasm bank at the FCA-UNNE and IBONE (CONICET-UNNE) and to tell about usual management practices and the procedures to preserve living plant material and the personnel's life involved in pre-pandemic and COVID-19 pandemic times. Teachers, researchers, undergraduate and graduate students carried out, for almost 40 years, the in vitro conservation of 56 cassava cultivars from different countries. Before March 2020, the bank management consisted mainly in scientific-technological activities for the conservation of the material and the search for parameters to establish an order of subcultures. Having decreed Social, Preventive and Compulsory Isolation in Argentina, conservation activities continued applying the usual practices by following political-institutional sanitary measures. To face the new sanitary scenarios, methodologies must be adjusted so that they are effective at maintaining viability of the plant material and at prolonging conservation time.
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Coronaviruses in the family Coronaviridae cause digestive and respiratory system infections in humans and animals. There are two subtypes of canine coronaviruses (CCoV), which are included in the alfacoronavirus, as CCoV I and CCoV II. CCoV-II is divided into two genotypes, CCoV-IIa and IIb. Although CCoV affects dogs of all ages and all diets, newborn puppies can be particularly susceptible and severely affected. According to the literature research, no molecular studies have been found in our country for the detection of canine coronavirus, especially in lower respiratory tract infections. In this study, it was aimed to detect and molecular characterization of CCoV un shelter dogs with lower respiratory tract infection. For this purpose, Bronchoalveolar Lavage (BAL) fluids taken from 40 shelter dogs with lower respiratory tract infections were examined. CCoV was detected in 3 of the BAL fluids of 40 dogs tested. A phylogenetic tree was constructed with the sequences obtained after the sequence analysis. It was determined that 2 of the 3 positive samples in the phylogenetic tree were CCoV-I and one sample was CCoV-II. In conclusion, this study revealed that CCoV-I and CCoV-II may play a role in lower respiratory system disorders of shelter dogs. In addition, the detection of two different CCoVs in different animals in the same shelter has been considered as an important data, and the detection of both types in dogs housed in crowded environments such as shelter conditions shows that the possibility of new variants or subtypes that may occur in the future should not be ignored.
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Coronavirus consists of single-stranded, enveloped and RNA virus, largest genome among all RNA viruses and has 4 proteins i.e. envelope, spike, nucleocapsid and membrane. Coronaviruses are classified into 4 genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Betacoronavirus most probably originated from bats and the virus may have jumped to avian species and evolved as Deltacoronavirus group. The avian coronaviruses jumped among other avian species, giving rise to Gammacoronavirus from Deltacoronavirus, while Betacoronavirus may have given rise to Alphacoronavirus. It is known that SARS-CoV-2 belongs to Betacoronavirus. This most similar virus is verified in bat and Malayan Pangolin. Analysis showed that SARS-CoV-2 most probably originated by recombination of both bat and pangolin viruses. Viral protein seroconversion and viral specific nucleotide positive documented in all COVID-19 patients tested provides confirmation of a link between the presence of this virus and the disease.
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Chicken infectious bronchitis (IB) is one of the epidemic diseases that cause severe economic loss to poultry industry and induced by the infection of infectious bronchitis virus (IBV). IBV was prone to mutation and recombination due to its genomic characteristics, resulting in new antigenic variants. In order to understand the prevalence and variation of IBV in Shanxi province and provide reference for IB epidemic prevention and control in this area, tissues of a broiler flock suspected of respiratory virus infection were collected in Jinzhong city, Shanxi province. Through PCR identification, chicken embryo inoculation, and sequencing verification, an IBV strain was isolated and named CK/Shanxi-01/2021. On the basis of S1 gene sequencing, the sequence was compared with those of the representative IBV strains of different genotypes deposited in NCBI database, and the phylogenetic tree was constructed. The results of genetic evolution analysis showed that the IBV strain isolated in this study was belonged to GI-19 genotype. The sequence alignment of CK/Shanxi-01/2021 with common IBV vaccine strains H120, M41, H52, 4/91, and LDT3-A showed that the nucleotide homology between the isolated strain and the current common vaccine strain was 78.1%-85.2%, and the amino acid homology was only 75.2%-78.4%. Compared with the sequences of GI-19 genotype strains, some new mutations, including V68I, S120A, A271T, N282T, and N291S, were identified in the S1 protein hypervariable region(HVR). Therefore, it is of great significance for the prevention and control of IB epidemic to strengthen the epidemiological monitoring of IBV and timely grasp the current epidemic IBV genotype and its variation characteristics.