Intracytoplasmic Inclusion Bodies Research Articles

Eptesipox virus-associated lesions in naturally infected big brown bats.

Bats have many unique qualities amongst mammals; one of particular importance is their reported tolerance to viruses without developing disease. Here, the authors present evidence to the contrary by describing and demonstrating viral nucleic acids within lesions from eptesipox virus (EfPV) infection in big brown bats. One hundred and thirty bats submitted for necropsy from Saskatchewan, Canada, between 2017 and 2021 were screened for EfPV by polymerase chain reaction (PCR); 2 had amplifiable poxvirus DNA. The lesions associated with infection were oral and pharyngeal ulcerations and joint swelling in 2/2 and 1/2 cases, respectively. These changes were nonspecific for poxvirus infection, although intracytoplasmic viral inclusion bodies within the epithelium, as observed in 2/2 bats, are diagnostic when present. Viral nucleic acids, detected by in situ hybridization (ISH), were observed in the epithelium adjacent to ulcerative lesions from both cases and within the joint proliferation of 1 case. A new isolate of EfPV was obtained from 1 case and its identity was confirmed with electron microscopy and whole genome sequencing. Juxtanuclear replication factories were observed in most cells; however, rare intranuclear virus particles were also observed. The significance of the presence of virus particles within the nucleus is uncertain. Whole genome assembly indicated that the nucleotide sequence of the genome of this EfPV isolate was 99.7% identical to a previous isolate from big brown bats in Washington, USA between 2009 and 2011. This work demonstrates that bats are not resistant to the development of disease with viral infections and raises questions about the dogma of poxvirus intracytoplasmic replication.

Clinical and Molecular Detections of Lumpy Skin Disease: Possibilities of Co-infection with Foot-and-Mouth Disease

Lumpy skin disease (LSD) and foot and mouth disease (FMD) are notable viral diseases of cattle. This report aimed to highlight the possibilities of an uncommon case of LSD and FMD co-infection. The report also presents the clinical and molecular detection of LSD virus in six crossbred calves and LSD and FMD virus co-infection in a heifer at small-scale dairy farms located in northern Ethiopia. Nasal swabs and tissue samples were collected following aseptic techniques from the six calves suspected of having LSD and a tissue sample from one heifer suspected of having LSD-FMD co-infection and submitted to the laboratory for cell culture and real-time polymerase chain reaction (PCR) tests. Different-sized, firm, painful skin nodules with necrotic centers were seen on different parts of the calves’ body. Swelling of the prescapular and prefemoral lymph nodes, conjunctivitis, and corneal cloudiness were also observed. Uniquely, one heifer was seen with erosive lesions in the oral cavity and tongue, salivation, lameness, and skin nodules. Intracytoplasmic inclusion bodies, a distinctive feature of LSD virus, and the formation of syncytia, a characteristic of FMD virus, were observed in the cell lines. The heifer was diagnosed with a rare co-infection of LSDV and FMDV based on clinical signs, cell culture, and real-time PCR test results. The other six calves were diagnosed with the LSD virus. Treatment with broad-spectrum antibiotics, local wound cleansing, and anti-inflammatory drugs was initiated. Unfortunately, the heifer with LSD-FMD co-infection died while under treatment, and just three calves with LSD were recovered. It can be concluded that vaccination of animals against both diseases and promotion of bio-security protocols in farms is more helpful than treatment, and early case reporting is also warranted to avoid losses related to the diseases.

Infant glycogen storage disease type Ⅳ: a clinicopathological and genetic characteristics analysis of five cases

Objective: To investigate the clinical pathology and gene mutation characteristics of patients with glycogen storage disease type Ⅳ (GSD Ⅳ). Methods: The clinical data, liver histopathology and ultrastructural morphology, and gene sequencing results of 5 GSD Ⅳ cases diagnosed in the Children's Hospital Affiliated to Shanghai Jiaotong University School of Medicine and the Children's Hospital of Fudan University from January 2015 to February 2022 were collected and analyzed retrospectively. Results: Among the 5 cases, 3 were male and 2 were female, ranging in age from 4 months to 1 year and 9 months. The clinical manifestations included fever, hepatosplenomegaly, liver insufficiency, growth retardation and hypotonia. Four cases had liver biopsy showing ground-glass-like changes in hepatocytes with intracytoplasmic inclusion bodies and varying degrees of fibrosis. Liver electron microscopy in 2 cases showed that the level of glycogen increased to varying degrees, and the cytoplasm was filled with low electron density substances. Genetic testing revealed that 3 cases had compound heterozygous variants in GBE1 gene; 1 case had a single pathogenic variant in GBE1 gene; and 1 case was deceased with no genetic testing, but each parent was tested for a heterozygous variant in the GBE1 gene. A total of 9 GBE1 gene mutations were detected, 3 of which were reported mutations and 6 novel mutations. One case died of liver cirrhosis, and 1 case underwent autologous liver transplantation. After transplantation, the liver function basically returned to normal, and the growth and development improved; the other 3 cases were managed through diet control and symptomatic treatment. Conclusions: CSD Ⅳ is an extremely rare inherited metabolic disease caused by GBE1 gene mutation, often presenting with hepatic and neuromuscular disorders, with heterogeneous clinical manifestations. The diagnosis mainly depends on histopathology and a pedigree gene analysis.

A Multiplex RT-PCR Method for the Detection of Reptarenavirus Infection.

Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), a fatal disease of boid snakes with an economic and ecological impact, as it affects both captive and wild constrictor snakes. The clinical picture of BIBD is highly variable but often only limited. Intracytoplasmic inclusion bodies (IB), which develop in most cell types including blood cells, are the pathognomonic hallmark of BIBD; their detection represents the diagnostic gold standard of the disease. However, IBs are not consistently present in clinically healthy reptarenavirus carriers, which can, if undetected, lead to and maintain the spread of the disease within and between snake populations. Sensitive viral detection tools are required for screening and control purposes; however, the genetic diversity of reptarenaviruses hampers the reverse transcription (RT) PCR-based diagnostics. Here, we describe a multiplex RT-PCR approach for the molecular diagnosis of reptarenavirus infection in blood samples. The method allows the detection of a wide range of reptarenaviruses with the detection limit reaching 40 copies per microliter of blood. Using 245 blood samples with a reference RT-PCR result, we show that the technique performs as well as the segment-specific RT-PCRs in our earlier studies. It can identify virus carriers and serve to limit reptarenavirus spreading in captive snake collections.

Species differences in small intestinal exposure-related epithelial vacuolation in rats and dogs treated with a heteroaryldihydropyrimidine molecule.

Small intestinal epithelial vacuolation induced by a heteroaryldihydropyrimidine compound (HAP-1) was observed in rats but not in dogs at termination in screening toxicity studies, despite the plasma exposure being higher in dogs. To understand the species differences, investigational studies with multiple time points following single dose (SD) and 7-day repeated dose (RD) were conducted in both species at doses resulting in comparable plasma exposures. In rats, epithelial vacuolation in the duodenum and jejunum were observed at all time points. In dogs, transient vacuolation was noted at 8h post-SD (SD_8h) and 4h post-RD (RD_4 h), but not at termination (RD_24 h). Special stains demonstrated lipid accumulation within enterocytes in both species and intracytoplasmic inclusion bodies in rats. Transmission electron microscopy identified these inclusion bodies as endoplasmic reticulum (ER) membranous structures. Transcriptomic analysis on jejunal mucosa at SD_8 h and RD_24 h revealed perturbations of lipid metabolism-related genes at SD_8 h in both species, but not at RD_24 h in dogs. ER stress-related gene changes at both time points were observed in rats only. Despite comparable HAP-1 plasma exposures, the duodenum and jejunum tissue concentrations of HAP-1 and acyl glucuronide metabolite were >5- and >30-fold higher in rats than in dogs, respectively. In vitro, similar cytotoxicity was observed in rat and dog duodenal organoids treated with HAP-1. In conclusion, HAP-1-induced intestinal epithelial vacuolation was related to lipid metabolism dysregulation in both species and ER-related injuries in rats only. The species differences were likely related to the difference in intestinal exposure to HAP-1 and its reactive metabolite.

Viral skin diseases in odontocete cetaceans: gross, histopathological, and molecular characterization of selected pathogens.

Fifty-five skin lesions from 31 stranded cetaceans along the Canary coasts (2011-2021) were submitted to macroscopic, histological, and molecular analyses to confirm infection by cetacean poxvirus, herpesvirus and cetacean morbillivirus. They were macroscopically categorized into eight categories with respective subcategories according to their color, shape, size, and consistency. Cetacean poxvirus was detected in 54.54% of the skin lesions through real-time and conventional PCRs based on the DNA polymerase gene. Additionally, herpesvirus and morbillivirus were currently detected from 43.63 and 1.82% of the cutaneous lesions, respectively. Coinfection of poxvirus and herpesvirus was detected in nine of them (16.36%), which makes the present study the first to report coinfection by both pathogens in skin lesions in cetaceans. A plausible approach to histopathological characterization of poxvirus-and herpesvirus-positive skin lesions was established. Hyperkeratosis, acanthosis, ballooning degeneration, and intracytoplasmic inclusion bodies in vacuolized keratinocytes through the stratum spinosum were common findings in poxvirus skin lesions. Alphaherpesvirus was associated with a prominent acanthotic epidermis, moderate necrosis, multifocal dyskeratosis, and irregular keratinocytes with both cellular and nuclei pleomorphism. The common histopathological findings of both pathogens were observed in coinfection lesions. However, those associated with herpesvirus were considerably more remarkable. Relationships between molecular and microscopic findings were observed for the lesions that showed tattoo-like and tortuous patterns. Further multidisciplinary diagnostic studies of infected skin lesions are needed to understand the epidemiology of these emerging infectious diseases.

Fatal avian pox in a backyard flock of domestic ducks in Bangladesh

AbstractThis case study describes a fatal avian pox outbreak in a backyard flock of domestic ducks (Anas platyrhynchos domesticus) in Bangladesh. A total of six ducks of varying ages were affected and presented with solid tumour‐like masses on the legs. A 6.5‐month‐old female duck was presented for clinical examination, and a 2 cm3 solid, nodular mass was surgically excised from the left leg. Histopathological examination of the tumour mass revealed marked epidermal hyperplasia with ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies, as well as severe proliferation and necrotising dermatitis, with marked heterophilic and histiocytic infiltration. Electron microscopy revealed numerous dumbbell‐shaped virus particles within the intracytoplasmic inclusion bodies on degenerating keratinocytes, confirming Avipoxvirus infection. This is the first case report of avian pox in ducks in Bangladesh.

Clinicopathological Investigations Among Recurrent Camelpox Outbreaks in Omanis’ Arabian Camels (Camelus dromedarius)

Camelpox remains a widespread viral disease in camelids, with socioeconomic relevance. The present study explored the hematological, biochemical, and histopathological alterations in dromedary-racing camels from the North of Oman infected with camelpox virus diagnosed by real-time PCR. Blood and skin samples were collected from camels with clinical signs and skin lesions (n=4) and from healthy camels (n=3) from 10 different camel herds. The results indicated that the infected camels showed clinical signs, including pyrexia, lacrimation, nasal mucus discharge, affixed and swollen eyelids, emaciation, and pimples on the skin of the head, legs, and abdomen. Hemoglobin, hematocrit, and platelets were significantly greater, with a significant reduction in leukocyte and lymphocyte counts in infected camels than in healthy camels. Infected camels had higher CK and creatinine levels and hepatic-related metabolites, including AST, ALP, AST, GGT, and LDH, than the apparently healthy camels. Histopathological examination of skin scab samples revealed ballooning degeneration of epidermal cells in the presence of typical large eosinophilic intracytoplasmic inclusion bodies and suppurative dermatitis following secondary bacterial infection in all examined infected camels. Camelpox viral DNA was detected using real-time PCR in the blood and skin samples of all infected camels. These findings in dromedary-racing camels associated with a molecular diagnosis of camelpox are described for the first time in the Sultanate of Oman. Therefore, further studies are warranted.

Pathological and molecular investigation of canine distemper virus: Phylogenetic analysis of co-circulating genetic lineages in Türkiye.

Canine distemper virus (CDV) is a highly contagious virus that infects a wide variety of animals of carnivore species and may cause manifestations from subclinical infection to fatal disease. In this study, dogs clinically suspected having distemper were examined by reverse transcriptase-polymerase chain reaction (RT-PCR), histopathology and immuno-histochemistry. By histopathological examination, characteristic intracytoplasmic and/or intranuclear inclusion bodies were observed in the lung, stomach, small intestine, liver, kidney, spleen and central nervous system. Interstitial and broncho-interstitial pneumonia, gastroenteritis and encephalitis were revealed. CDV antigens were detected in all tissues with characteristic histopathological findings. The antigens were more abundant in the bronchial andbronchiolar epithelium and in the syntitial cells. Phylogenetic analyses were performed using the PCR-amplified partial sequences of the genes encoding the viral heamagglutinin and fusion proteins. The phylogenetic trees showed that the newly determined sequences were diverse and clustered within different lineages of the European or the Arctic strains.

Meningoencephalitis caused by concurrent infection with canine distemper virus and a unique Sarcocystis sp. in a gray fox.

A deceased 9-wk-old male gray fox (Urocyon cinereoargenteus) with a history of decreased ambulation and diarrhea was submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory. No significant gross findings were evident on postmortem examination. Histologically, the cerebrum and brainstem had mild necrotizing meningoencephalitis with protozoal schizonts and merozoites. Additionally, glial cells contained intracytoplasmic and intranuclear viral inclusion bodies. Sections of the cerebrum were positive for canine distemper virus (CDV) and negative for Sarcocystis neurona on immunohistochemistry. Bayesian analysis revealed that this Sarcocystis sp. clustered most closely with a clade of unnamed Sarcocystis sp. found in viperid snakes, with a posterior probability of 99%. CDV likely played a significant role in the expression of clinical sarcocystosis in this gray fox.

First report of Psittacid Avipoxvirus in Agapornis in Mexico

A postmortem study was performed on two lovebirds (Agapornis fischeri and Agapornis personatus) that had scabs in the periocular region and on the eyelid, as well as serous blepharitis. Microscopically, the eyelids showed ulcers, necrosis and serocellular crusts, severe hyperplasia of keratinocytes with eosinophilic intracytoplasmic inclusion bodies (Bollinger's bodies), bacterial colonies of gram-positive coccoid morphology and PAS-positive septate and 45° branching hyphae. The microbiological study identified the colonies as Staphylococcus spp. and Aspergillus fumigatus, respectively. Using molecular techniques, avian pox clade C was identified on the eyelid. This is the first report in Mexico of a case of avian pox in parrots associated with clade C Avipoxvirus

Brazilian porcupinepox virus infection in a free-ranging neotropical porcupine in Mato Grosso, Brazil.

Although the novel Brazilian porcupinepox virus (BPoPV) can infect wild porcupines, its lethality and zoonotic potential are not well-established. In May 2021, a free-ranging neotropical porcupine (Coendou longicaudatus boliviensis) rescued from the natural savanna in the Brazilian Cerrado, Mato Grosso State, was presented with a lethal poxviral infection. Clinical signs and PCR detection of BPoPV supported the diagnosis. Poxviral lesions included erythema, exfoliative dermatitis, and erosions involving mainly the face, hindlimb, and vulva. Histologically, the lesions consisted of proliferative and necrotic dermatitis, intraepidermal and follicular pustules, and intracytoplasmic inclusion bodies in keratinocytes. Phylogenetic analysis revealed BPoPV strains closely related to other rodent-infecting poxviruses. This poxviral infection resulted in the death of a specimen of C. l. boliviensis; the effect on species conservation, and the potential of spillover into humans and other vertebrates remain unknown.

A lower motor neuron disease in takahē (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease

Aims: To investigate the pathogenesis of a disease in takahē with intracytoplasmic inclusion bodies in lower motor neurons. Methods: Four cases aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the bird with spinal dysfunction were examined by electron microscopy. Results: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D so they were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method. A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. Conclusions: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Lesions and viral loads in racing pigeons naturally coinfected with pigeon circovirus and columbid alphaherpesvirus 1 in Australia.

Columbid alphaherpesvirus 1 (CoHV1) is associated with oral or upper respiratory tract lesions, encephalitis, and occasional fatal systemic disease in naive or immunosuppressed pigeons. Clinical disease is often reported with CoHV1 and coinfecting viruses, including pigeon circovirus (PiCV), which may cause host immunosuppression and augment lesion development. A natural outbreak of CoHV1 and PiCV coinfection occurred in a flock of 60 racing rock pigeons (Columba livia), in which 4 pigeons succumbed within 7 d of clinical onset. Lesions included suppurative stomatitis, pharyngitis, cloacitis, meningitis, and tympanitis, with eosinophilic intranuclear inclusion bodies consistent with herpesviral infection. In addition, large numbers of botryoid intracytoplasmic inclusion bodies were present in the skin, oral mucosa, and bursa of Fabricius, suggestive of circoviral infection, which was confirmed by immunohistochemistry. The concurrent viral load of CoHV1 and PiCV was high in liver, oropharynx, and bursa of Fabricius. We found PiCV in oro-cloacal swabs from 44 of 46 additional birds of variable clinical status, PiCV alone in 23 birds, and coinfection with CoHV1 in 21 birds. Viral copy numbers were significantly higher (p < 0.0001) for both viruses in clinically affected pigeons than in subclinical qPCR-positive birds. The CoHV1-induced lesions might have been exacerbated by concomitant PiCV infection.

Disease development in red hybrid tilapia following single and co-infection with tilapia lake virus and Streptococcus agalactiae

Tilapia lake virus (TiLV) has recently been reported to cause infection in cultured and wild tilapia globally, and co-infection between TiLV and other bacterial pathogens contributes to higher mortality and economic losses. Understanding the pathology and pathogenesis of the co-infection could help in controlling the outbreaks. This study determines and compares the severity of single and co-infection of TiLV and Streptococcus agalactiae in red hybrid tilapia. The tilapias were challenged intra-peritoneal with 106 TCID50/mL of TiLV and 103 CFU/mL of S. agalactiae either as single or as co-infection. Fish were divided into five experimental groups with four replicates (30 fish/replicate). Group 1 received a single TiLV intra-peritoneal infection at 106 TCID50/mL of TiLV, Group 2 received a single S. agalactiae intra-peritoneal infection at 103 CFU/mL of S. agalactiae, Group 3 was co-infected intra-peritoneal with TiLV and S. agalactiae, Group 4 was co-infected with S. agalactiae and TiLV, and Group 5 was the control unchallenged. Co-infection by S. agalactiae in Group 3 was done at 13-days post infection (dpi), while co-infection by TiLV in Group 4 was at 2-dpi. Following challenge, fish were monitored daily for 20 days, while samples of brain and liver were collected at 2-dpi intervals for viral and bacterial load analyses. Co-infected fish recorded higher cumulative mortalities (60% for TiLV-S. agalactiae and 73% for S. agalactiae-TiLV) compared to single infection (40% for TiLV and 20% for S. agalactiae). Both single and co-infected fish showed similar clinical signs and gross lesions, respective to the exposed pathogen. Notable histopathological changes included the presence of multinucleated syncytial giant cells and intracytoplasmic inclusion bodies following TiLV infection either in single or co-infection, but not in S. agalactiae single infection. The mean of histopathological score for brain and liver, and pathogen loads were significantly (p < 0.05) higher in the co-infected fish. This study describes the disease development following TiLV and S. agalactiae co-infection and concluded that co-infection of TiLV and S. agalactiae increases the mortality and enhances the disease severity in tilapia.

Identification and characterisation of lumpy skin disease virus recently isolated from Giza, Egypt

Lumpy skin disease (LSD) is a viral disease, geographically distributed in Africa and now, vigorously spread in the Near East and also in Europe and Asia. It has a significant economic impact on cattle industry in Africa. The aim of this study was isolation and rapid identification of LSD virus circula­ting in Egypt from clinically suspected cattle based on clinical and molecular basis in a rapid and accurate way. Fifteen representative specimens (skin sitfasts) were collected in 2018 from clinically infected cattle in Giza governorate, Egypt. The virus was isolated on chorioallantoic membrane of specific pathogen free embryonated chicken eggs and Madin Darby Bovine Kidney tissue culture cells. The isolated virus was identified and confirmed by conventional polymerase chain reaction (PCR) and real-time PCR. Histopathological examination of the lesions showed a pathognomic intracytoplasmic inclusion body in dermal stroma section. The section of dermal layer revealed vasculitis with projection of its endothelial lining. It was concluded that LSD was enzootic in Egypt and still circulating among Egyptian cattle so that LSD virus could be isolated and identified by traditional and molecular diagnostic methodes. Real time PCR assay could be applied for rapid and accurate confirmation of the field isolate of LSD virus.

Lymphocystis Disease Virus Infection in Clownfish Amphiprion ocellaris and Amphiprion clarkii in Taiwan.

Lymphocystic disease affects over 150 species of marine and freshwater fish worldwide. In this study, the lymphocystis pathogen was found in 2 (Amphiprion ocellaris and Amphiprion clarkii) of the 9 species of clownfish. Detection of lymphocystis disease virus (LCDV) was based on histopathological study, electron microscope observation of virus particles and gene sequence analysis from the MCP region. Infected A. ocellaris hosts showed sparse, multifocal, white, stiff, papilloma-like nodules on the body, skin, gills and fins; while, on A. clarkia, nodules were found on the operculum skin. Histopathologic study showed lymphocystic cells with an irregular nucleus, enlarged cytoplasm and intracytoplasmic inclusion bodies surrounded by the cell membrane. The viral particle presents virions 180-230 nm in diameter, hexagonal in shape with an inner dense nucleoid under transmission electron micrographs (TEM). From the ML polygenetic tree, the clownfish LCVD genotype was closely related to the LCDV strain from paradise fish, Macropodus opercularis (KJ408271) (pairwise distance: 92.5%) from China, then followed by the strain from Spain (GU320726 and GU320736) (pairwise distance: 90.8-90.5%), Korea (AB299163, AB212999, AB213004, and AB299164) (pairwise distance: 91.5-80.5%) and lastly Canada (GU939626) (pairwise distance: 83%). This is the first report of lymphocystis disease in A. clarkii in Taiwan.

Identification, molecular characterization, and pathological features of orf virus in sheep and goats in Punjab province, Pakistan

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6-98.7% nucleotide and 97.6-100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.

Identification and pathogenetic study of tilapia lake virus (TiLV) isolated from naturally diseased tilapia

As an emerging pathogen, tilapia lake virus (TiLV) has caused severe socio-economic impacts and remains a devastating factor in wild and farmed tilapia. Early diagnosis and timely reporting of TiLV are very important and necessary. In the study, a TiLV infection event in a tilapia farm is studied and reported. Naturally diseased tilapia with clinical signs such as anorexia, exophthalmia, skin abrasion and hemorrhage are collected. Multiple tissue lesions of liver, spleen and kidney are observed by histopathological analysis, such as syncytial formation, intracytoplasmic inclusion body and necrosis. No bacterial pathogens are identified from liver, spleen and kidney of the diseased tilapia, while all of them show TiLV-positive. E-11 cells are employed for TiLV isolation, and cytopathic effects are developed following incubation with supernatants of the homogenized tissues collected from the diseased tilapia. Electron micrographs of the infected cells and purified TiLV show that nearly round-shaped, enveloped viral particles (60–80 nm) can be identified. Segment 1 of the isolated TiLV has a high sequence similarity to other isolates, but phylogenetic analysis shows that it is placed in a unique cluster. Experimental infection with the isolated TiLV can cause high mortality (> 90%) in healthy Nile tilapia, with the similar clinical signs and tissue lesions to those found in naturally infected tilapia. Moreover, vacuolation and necrosis in brain, inclusion body and necrosis in heart, megalocytes and necrosis in intestine are observed in experimentally infected tilapia. Muscle, spleen, liver, intestine, brain, heart, kidney and gill of the re-infected tilapia show TiLV-positive, and liver, spleen and brain are the main target tissues. The data so far confirm a TiLV-associated disease outbreak in China, enriching the relevant information of TiLV.

Graph theoretical network analysis, in silico exploration, and validation of bioactive compounds from Cynodon dactylon as potential neuroprotective agents against α-synuclein.

Introduction: Parkinson's disease (PD) is a chronic, devastating neurodegenerative disorder marked by the death of dopaminergic neurons in the midbrain's substantia nigra pars compacta (Snpc). In alpha-synuclein (α-Syn) self-aggregation, the existence of intracytoplasmic inclusion bodies called Lewy bodies (LBs) and Lewy neurites (LNs) causes PD, which is a cause of neuronal death. Methods: The present study is aimed at finding potential bioactive compounds from Cynodon dectylon that can degrade α-Syn aggregation in the brain, through in silico molecular docking investigations. Graph theoretical network analysis was used to identify the bioactive compounds that target α-Syn and decipher their network as a graph. From the data repository, twenty-nine bioactive chemicals from C. dactylon were chosen and their structures were retrieved from Pubchem. On the basis of their docking scores and binding energies, significant compounds were chosen for future investigation. The in silico prediction of chosen compounds, and their pharmacokinetic and physicochemical parameters were utilized to confirm their drug-likeness profile. Results: During molecular docking investigation the bioactive compounds vitexin (-7.3 kcal.mol-1) and homoorientin (-7.1 kcal.mol-1) showed significant binding energy against the α-Syn target protein. A computer investigation of molecular dynamics simulation study verifies the stability of the α-Syn-ligand complex. The intermolecular interactions assessed by the dynamic conditions indicate that the bioactive compound vitexin has the potency to prevent α-Syn aggregation. Conclusion: Interestingly, the observed results indicate that vitexin is a potential lead compound against α-Syn aggregation, and in vitro and in vivo studies are warranted to confirm the promising therapeutic capability.