Intracytoplasmic Viral Inclusions Research Articles

Epidemiologic, clinicopathologic, and diagnostic findings in pet rabbits with myxomatosis caused by the California MSW strain of myxoma virus: 11 cases (2022-2023).

To determine epidemiologic features of naturally occurring myxomatosis in domestic rabbits in California and to characterize clinicopathologic and diagnostic findings. 11 client-owned rabbits, Oryctolagus cuniculus subsp domesticus. A prospective study of pet rabbits with myxomatosis seen at an exotic animal specialty clinic in Santa Cruz county, California, was conducted between January 1, 2022, and December 31, 2023. Rabbits were included in the study if they had bilateral blepharedema and were PCR positive for myxoma virus. All infected rabbits had spent time outdoors. Common clinical signs included bilateral blepharedema (11/11), anogenital edema (10/11), rectal temperature ≥ 39.7 °C (5/9), and sudden death (4/11). Eyelid biopsies from all rabbits (11/11) were positive for myxoma virus by qualitative PCR followed by Sanger sequencing (100% nucleotide identity to strain MSW, also known as California/San Francisco 1950 [Genbank accession KF148065]). Most rabbits had keratinocytes containing eosinophilic intracytoplasmic viral inclusions in biopsies of edematous skin (8/11) and lymphocyte necrosis in the spleen (10/11). Immunohistochemistry identified myxoma virus in samples of skin, heart, lung, ileum, spleen, and lymph node. Clinical signs of myxomatosis caused by the MSW strain of myxoma virus are distinctive but subtle. Cases occur regularly in the Santa Cruz and San Jose regions of California. As infection with this virus is almost 100% fatal and no vaccine is available in the US, owners of domestic rabbits in endemic areas should keep their pets indoors or behind mosquito screens. Myxomatosis is a reportable disease in the US, and the appropriate state or federal agencies should be contacted when outbreaks occur.

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.

Comparative Histopathology of Bovine Acute Interstitial Pneumonia and Bovine Respiratory Syncytial Virus-Associated Interstitial Pneumonia

Acute interstitial pneumonia (AIP) is a significant disease of cattle and many aetiologies have been implicated on the basis of the characteristic pathological lesions. Bovine respiratory syncytial virus (BRSV) is one of the key aetiological factors in bovine respiratory disease complex and several studies have suggested, controversially, that BRSV may be an underlying cause of bovine AIP. BRSV infection is known to cause several distinctive histopathological changes, including epithelial syncytia formation and intracytoplasmic viral inclusions. However, distinguishing bovine AIP from BRSV-related pneumonia by clinical presentation, gross pathology or histopathology can sometimes be challenging. In order to identify the potential distinguishing features, we compared the histopathological findings of AIP that were, and were not, associated with BRSV infection in naturally occurring cases. We found that multinucleated giant cells were more frequently identified in cattle with AIP while bronchiolitis was more common in BRSV-infected cattle. However, this was not considered a sole indicator of either disease group. Statistically, we identified that a combination of several histopathological features, including alveolar septal necrosis, presence of multinucleated giant cells and bronchiolitis, can serve as an excellent indicator for distinguishing between idiopathic AIP and BRSV-related pneumonia, with a strong statistical significance (P=0.0004). Based on the results of this retrospective study, we present a histopathological scoring system for predicting BRSV-associated AIP.

Histologic lesions of experimental infection with Lymantria dispar multicapsid nucleopolyhedrovirus and Lymantria dispar cytoplasmic polyhedrosis virus in European gypsy moth caterpillars (Lymantria dispar dispar).

European gypsy moths (Lymantria dispar dispar) are an invasive species in North America, and are listed by the International Union for the Conservation of Nature as one of the 100 most destructive invasive species worldwide. They have several known viruses, some of which are used as biological control agents. However, there are no detailed descriptions of many entomopathogenic viral infections, including in European gypsy moths, using bright-field microscopy. In this study, 11 European gypsy moth caterpillars were evaluated histologically: 4 were experimentally infected with Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV; Baculoviridae); 4 were experimentally infected with Lymantria dispar cytoplasmic polyhedrosis virus (LdCPV; Reoviridae); 3 control animals were uninfected. A complete tissue set was evaluated in all animals from all groups using bright-field microscopy, including epidermis, cuticle, striated muscle, tracheae, foregut, midgut, hindgut, Malpighian tubules, hemocytes, fat body, and nervous system. LdMNPV-infected caterpillars had marked karyomegaly and intranuclear viral inclusions in cells of the epidermis, tracheae, fat body, and hemocytes. LdMNPV-infected caterpillars also had hyperplasia and hypertrophy of epidermal and tracheal epithelial cells. LdCPV-infected caterpillars had numerous granular eosinophilic intracytoplasmic viral inclusions in midgut epithelial cells. Both LdMNPV-infected and LdCPV-infected caterpillars had atrophy of fat body adipocytes; this change was more pronounced in LdCPV-infected caterpillars. This work provides the first detailed descriptions of these viral infections in European gypsy moth caterpillars using bright-field light microscopy and provides images of normal histology from control caterpillars.

CANINE DISTEMPER VIRUS IN THE SEA OTTER (ENHYDRA LUTRIS) POPULATION IN WASHINGTON STATE, USA.

Before 2001, all serosurveys for morbilliviruses in sea otters (Enhydra lutris) in California, Washington, and Alaska, US, documented a 0% seroprevalence. The first published serologic detections of morbillivirus in sea otters occurred in 2001-02 in live-captured Washington sea otters, with a documented 80% seroprevalence. We conducted a retrospective study of sea otter cases from 1989 to 2010 compiled at the US Geological Survey, National Wildlife Health Center to identify cases of morbilliviral disease in Washington sea otters and to characterize the disease using immunohistochemistry, reverse transcription (RT)-PCR, genetic sequencing, virus isolation, and serology. We identified six cases of morbilliviral disease and 12 cases of morbilliviral infection in this population of sea otters during 2000-10. Significant histologic findings included inflammation in the white and gray matter of the brain characterized by lymphoplasmacytic perivascular cuffing, neuronal necrosis, and satellitosis in gray matter and by spongiosis, myelin degeneration, spheroids, and gemistocytes in white matter. Intranuclear and intracytoplasmic viral inclusion bodies were found in neurons, Purkinje cells, and glia. Immunohistochemistry for canine distemper virus (CDV) showed positive staining in neurons, glial cells, and cell processes. A pan-morbillivirus RT-PCR with subsequent restriction endonuclease digestion or sequencing identified CDV. Virus isolation was not successful. Two sea otters with morbilliviral encephalitis showed greater antibody titers to CDV than phocine distemper virus. Histologic changes were confined to the central nervous system and resembled neurologic canine distemper in domestic dogs. Cases of sea otters with morbilliviral infection without histologic changes could represent early infections or incompletely cleared sublethal infections. We found that morbillivirus was present in the Washington sea otter population as early as 2000, and we provide a description of the pathology of canine distemper in sea otters.

Red sea bream iridovirus disease (RSIVD) outbreak in Asian seabass (Lates calcarifer) cultured in open estuarine cages along the west coast of India: First report

In the present study, moribund Asian seabass (Lates calcarifer) fish samples were collected from open estuarine cages and observed for clinical signs followed by molecular diagnosis using primers targeting DNA polymerase gene of RSIV reported from OIE. The 568 bp amplicon confirmed the 100% sequence similarity with the reported sequences of RSIV from other countries. It formed the four clusters (RSIV, ISKNV, TBIV, and SDDV) with showing the close relation to Korean RSIV isolate than the RSIV isolate of Japanin the phylogenetic tree and multiple alignment analysis. The histopathogical examinations revealed, necrosed cellular material, melanomacrophage centers, RBC proliferation, lymphocytic infiltration, increased vacuoles and irregular intracytoplasmic viral inclusion bodies were observed in infected fish gill and spleen tissues. The challenge test with tissue homogenate reproduced 100% mortality in the healthy seabass by 6th day post infection (dpi). The first report of the virus in cultured Asian seabass from the region has been notified to OIE.

Peste des petitis ruminants morbillivirus infection in lambs and young goats at Qassim region, Saudi Arabia

An outbreak of peste des petits ruminants (PPR) in lambs and young goats of Najdi breed of sheep and goats occurred during winter 2005 at Qassim region of central Saudi Arabia. The PPR infection was confirmed by demonstration of antibodies against the virus in the serum of clinically-ill young sheep and goats using competitive ELISA test. Clinical examination of infected animals showed fever, salivation, lacrimation, mucopurulent nasal discharge, difficult breathing and diarrhoea. The disease was particularly severe in the goats. Morbidity was about 20% and mortality was less than 3 percent. Autopsy showed necrotic and ulcerative lesions in the mouth, stomach and intestine. Mesenteric lymph nodes were swollen and congested. The lungs were patchy pneumonic mostly at the diaphragmatic and apical lobes. Liver and kidney lesions were seen in goats only and both organs were congested and necrotic. Histopathological examination revealed necrotic inflammation in the gastrointestinal tract. Intracytoplasmic viral inclusions were seen in the enterocytes of goats. Lung sections showed bronchopneumonia and syncytial and giant cells. The bronchial epithelium of goats had intracytoplasmic viral inclusions. Extensive coagulation necrosis, fatty degeneration and presence of intracytoplasmic viral inclusions were seen in hepatocytes and syncytial cells were evident in biliary epithelium of goats. Congestion, coagulation necrosis and syncytial cells were seen in the renal tubular epithelium of goats only. In a survey to determine the magnitude of the outbreak, PPR antibodies were evidenced in 363/996 (36.6%) sheep and 530/962 (55.1%) goats.

Frog Virus 3 Infection, Cultured American Bullfrogs

Frog Virus 3 Infection, Cultured American Bullfrogs

Cytomegalovirus enterocolitis complicated by perforated appendicitis in a premature infant

A 9-week-old, former 30-week estimated gestational age premature infant had recurrent episodes of abdominal distention. Laparotomy revealed partial small bowel obstruction caused by ileocecal inflammation with stenosis, and a perforated appendix with fistulization into the cecum. The resected appendix and ileocecal junction showed intranuclear and intracytoplasmic viral inclusions, and were cytomegalovirus positive by immunoperoxidase staining, which implicated cytomegalovirus as the etiology of the recurring bouts of enterocolitis with appendicitis. Cytomegalovirus is frequently overlooked in the differential diagnosis of enterocolitis and chronic gastrointestinal symptoms in infants, but should be included as in older immunocompromised patients.

Cytomegalovirus colitis: a rare cause of massive lower GI bleed in an immunocompetent patient

A 70 year-old white female was admitted to our institution for right hip disarticulation due to severe peripheral vascular disease. Her past medical history was significant for Hypertension, GERD, Coronary artery disease and peripheral vascular disease. She was on long term aspirin therapy. After the hip surgery, patient was placed on Warfarin. On post-operative day 6, patient developed massive lower gastrointestinal bleeding with passage of bright red blood per rectum. This was assoociated with severe hypotension with systolic blood pressue around 70 mm Hg. Laboratoty data showed hemoglobin of 7.9 gm%, a drop of about 4 grams since admission. Patient also had INR of 3.8 which was reversed with Vitamin K injections and fresh frozen plasma infusions. Even correction of coagulopathy did not result in slowing of lower GI bleeding. An emergent Tc-99m labelled RBC scan was performed which showed increased localization of tracer in the rectum, this was followed by an angiogram which failed to reveal the site of bleeding. An emergent surgery (left hemicolectomy/rectosigmoid resection) was considered due to continued bleeding but patient declined to undergo any surgical intervention. After hemodynamic stabalization, colonoscopy was performed which showed numerous discrete ulcers scattered throughout the colon including rectum. Biopsies obtained from these ulcers showed areas of ulceration with acute inflammation and granulation tissue formation. Focally, within the lamina propria, there were occassional cells with marked cytomegaly and smudged chromatin with some intracytoplasmic viral inclusions. The tissue was negative immunohistochemically for antigens of herpes simplex virus type 1 and type 2. An immunohistochemical stain for cytomegalovirus antigen shows numerous immunohistochemically positive cells within the lamina propria, both within areas of ulceration and in some areas of otherwise unremarkable mucosa. Patient was subsequently treated with Gancyclovir with resolution of her symptoms. Our patient did not have any other underlying immunocompromising illness which could have predisposed her to CMV colitis. Our case demonstates the importance of considering rare infectious causes of GI bleed in immunocompetent persons which could otherwise prove fatal.

Severe Acute Respiratory Syndrome: The Evolution of a New Epidemic

Severe Acute Respiratory Syndrome: The Evolution of a New Epidemic

An epizootic of fibromatosis in gray squirrels (Sciurus carolinensis) in Florida.

Beginning in the fall of 1998 and extending into the spring and early summer of 1999 there was a large epizootic of squirrel fibromatosis in squirrels in seven counties in peninsular Florida. Hundreds of gray squirrels (Sciurus carolinensis) with multiple cutaneous tumors were submitted or reported to biologists, veterinary hospitals, and private wildlife rehabilitators. Most squirrels died or were euthanized soon after submission. Twenty squirrels were submitted for necropsy. The majority of the squirrels examined were adults (12/20) and male (15/20). The number and location of tumors varied widely among the affected squirrels; however, a consistent finding was involvement of the eyelids (20/20). Histopathology revealed a proliferative population of mesenchymal cells within the dermis and marked ballooning degeneration of keratinocytes in the overlying epidermis. Intracytoplasmic viral inclusions were present in the neoplastic mesenchymal cell population and the degenerating keratinocytes. Ulceration and necrosis of the surface of the tumors or associated tissues was present in 14 of the 20 squirrels. Virions consistent with poxvirus were observed via electron microscopy in samples collected from a representative tumor. Death of the squirrels was attributed to emaciation, tissue damage, and severe negative energy balance associated with poxvirus infection and massive tumor growth. The underlying cause of this unusual epizootic of fibromatosis in gray squirrels remains unknown.

Detection of Porcine Circovirus 2 (PCV-2) DNA by Nested PCR from Formalin-Fixed Tissues of Post-Weaning Multisystemic Wasting Syndrome (PMWS) Pigs in Thailand.

A retrospective study for the detection of porcine circovirus 2 (PCV-2) DNA was conducted by nested PCR method in 16 cases of swine post-weaning multisystemic wasting syndrome (PMWS) in Thailand. Histopathology showed characteristic lesions of PMWS and intracytoplasmic viral inclusion bodies in macrophages infiltrating in lymphoid tissues. PCV-2 DNA was detected from formalin-fixed and/or formalin-fixed paraffin-embedded tissues from all pigs with PMWS. The amplified products were digested with Hae III.

Apoptosis Induced In Vitro and In Vivo During Infection by Ebola and Marburg Viruses

Induction of apoptosis has been documented during infection with a number of different viruses. In this study, we used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling to investigate the effects of Ebola and Marburg viruses on apoptosis of different cell populations during in vitro and in vivo infections. Tissues from 18 filovirus-infected nonhuman primates killed in extremis were evaluated. Apoptotic lymphocytes were seen in all tissues examined. Filoviral replication occurred in cells of the mononuclear phagocyte system and other well-documented cellular targets by TEM and immunohistochemistry, but there was no evidence of replication in lymphocytes. With the exception of intracytoplasmic viral inclusions, filovirus-infected cells were morphologically normal or necrotic, but did not exhibit ultrastructural changes characteristic of apoptosis. In lymph nodes, filoviral antigen was co-localized with apoptotic lymphocytes. Examination of cell populations in lymph nodes showed increased numbers of macrophages and concomitant depletion of CD8+ T cells and plasma cells in filovirus-infected animals. This depletion was particularly striking in animals infected with the Zaire subtype of Ebola virus. In addition, apoptosis was demonstrated in vitro in lymphocytes of filovirus-infected human peripheral blood mononuclear cells by TEM. These findings suggest that lymphopenia and lymphoid depletion associated with filoviral infections result from lymphocyte apoptosis induced by a number of factors that may include release of various chemical mediators from filovirus-infected or activated cells, damage to the fibroblastic reticular cell conduit system, and possibly stimulation by a viral protein.

Respiratory disease due to parainfluenza virus in adult bone marrow transplant recipients.

We reviewed the frequency and clinical course of parainfluenza virus (PIV) infections in 1,173 adult bone marrow transplant (BMT) recipients cared for at The University of Texas M.D. Anderson Cancer Center (Houston). Between January 1991 and September 1994, PIV was isolated from the respiratory secretions of 61 (5.2%) of these patients. Thirty-four (56%) of the 61 patients had uncomplicated upper respiratory tract illnesses and survived. The remaining 27 patients (44%) developed pneumonia, and the associated mortality was 37% (10 of 27 patients). Twenty-three (85%) of the patients with pneumonia had had preceding upper respiratory illnesses. Of the 10 patients who died, nine died within 100 days after transplantation. Histopathologic examination of lung tissue from seven patients revealed intracytoplasmic viral inclusions in six, a finding consistent with invasive PIV pneumonia, and viral changes in the seventh patient. Seven of the 10 patients who died had other serious concurrent infections. Of 42 patients who developed PIV infection early after transplantation (i.e., < 100 days), the frequency of pneumonia was higher among the 18 allogeneic BMT recipients (61%) than among the 24 autologous BMT recipients (42%), and the associated mortality was also higher (55% vs. 30%, respectively). PIVs are an important cause of life-threatening pneumonia in adult BMT recipients, particularly patients who have recently undergone allogeneic bone marrow transplantation.

Differentiation of filoviruses by electron microscopy

Cultured monolayers of MA-104, Vero 76, SW-13, and DBS-FRhL-2 cells were infected with Marburg (MBG), Ebola-Sudan (EBO-S), Ebola-Zaire (EBO-Z), and Ebola-Reston (EBO-R) viruses (Filoviridae, Filovirus) and examined by electron microscopy to provide ultrastructural details of morphology and morphogenesis of these potential human pathogens. Replication of each filovirus was seen in all cell systems employed. Filoviral particles appeared to enter host cells by endocytosis. Filoviruses showed a similar progression of morphogenic events, from the appearance of nascent intracytoplasmic viral inclusions to formation of mature virions budded through plasma membranes, regardless of serotype or host cell. However, ultrastructural differences were demonstrated between MBG and other filoviruses. MBG virions recovered from culture fluids were uniformly shorter in mean unit length than EBO-S, EBO-Z, or EBO-R particles. Examination of filovirus-infected cells revealed that intermediate MBG inclusions were morphologically distinct from EBO-S, EBO-Z, and EBO-R inclusions. No structural difference of viral inclusion material was observed among EBO-S, EBO-Z, and EBO-R. Immunoelectron microscopy showed that the filoviral matrix protein (VP40) and nucleoprotein (NP) accumulated in EBO-Z inclusions, and were closely associated during viral morphogenesis. These details facilitate the efficient and definitive diagnosis of filoviral infections by electron microscopy.

APPARENT CYTOMEGALOVIRUS EPITHELIAL KERATITIS IN A CARDIAC TRANSPLANT RECIPIENT

A 44-year-old immunosuppressed man developed initial symptoms of intermittent irritation of the left eye three months after cardiac transplantation. Symptoms increased, with decreased vision, photophobia, and lacrimation. Slit lamp examination showed slightly raised, swollen, grayish epithelium in a broad multibranching dendritic pattern associated with fine and medium punctate epithelial erosions that stained slightly with fluorescein. Histopathologic study of the corneal epithelial scraping demonstrated swollen epithelial cells with intranuclear and intracytoplasmic viral inclusions. Viral cultures manifested a cytopathic pattern characteristic of cytomegalovirus 14 days after inoculation on human embryonic lung cells (MRC-5). Pretransplantation cytomegalovirus IgM and IgG serologic titers were negative (less than 1:16 for IgG, no IgM noted) until the onset of symptoms. Subsequently, IgM titers rose against cytomegalovirus consistent with concurrent infection.

Frequency and diagnostic value of the virus-like filamentous intranuclear inclusions in giant cell tumor of bone, not associated with Paget's disease

This paper deals with the paramyxovirus-like intranuclear inclusions observed in giant cells tumours of bone (GCTB). Twenty-one (49%) of 43 cases of GCTB (1977-1985), either fresh and/or cultured, show these ultrastructural inclusions. Fifty samples of various bone lesions in which giant cell lesions occurred, including aneurysmal cysts, hyperparathyroidism, osteoblastoma, human and rat osteopetrosis, GCT of tendon sheaths, and non skeletal granuloma were used as controls. These, together with 20 samples of normal bone (osteoclasts) did not contain intranuclear or intracytoplasmic viral inclusions.

Mechanism of rabies virus entry into CER cells.

The early steps of rabies virus (CVS) infection in vitro were studied in chicken embryo-related (CER) cells. The infection was monitored by looking for specific intracytoplasmic viral inclusions using anti-rabies fluorescein isothiocyanate at 24 h after the addition of virus. The attachment of rabies virus to CER cells was shown to be inhibited by pretreatment of the cells with neuraminidase. These cells recovered their susceptibility to rabies virus infection 6 h after removal of the enzyme. Treatment of CER cells with neuraminidase after the viral attachment step did not inhibit infection. The subsequent delivery of infectious virions into acid prelysosomal vacuoles or lysosomes was studied using lysosomotropic agents. Ammonium chloride and chloroquine were used to prevent the virus fusion step thus preventing infection. Both drugs were shown to inhibit the early steps of infection, NH4Cl having a much earlier effect than chloroquine. The two drugs had no effect on the attachment step nor did NH4Cl inhibit virus multiplication. The use of metabolic inhibitors (2-deoxy-D-glucose and sodium azide) shows that the entry of rabies virus into CER cells does not require the involvement of cellular energy processes. In electron microscopy studies, the presence of rabies virus particles was detected in coated pits and coated vesicles as well as in uncoated vesicles, and later in lysosomes. These data indicate that the mechanism by which rabies virus enters CER cells is probably through adsorptive endocytosis and does not require the participation of cellular metabolic active processes.

Ultrastructural development and persistence of guinea pig cytomegalovirus in duet cells of guinea pig submaxillary gland.

Salivary glands from Hartley guinea pigs were experimentally infected with guinea pig cytomegalovirus (GPCMV) and examined by light and electron microscopy at different time intervals. Characteristic intranuclear and intracytoplasmic viral inclusions were observed in duct cells of infected animals. Viral inclusion counts and infectivity titers in the salivary gland reached maximum levels by 3 to 4 weeks after infection; infectivity persisted, though at reduced levels, for at least 30 weeks. Electron microscopic examination of viral inclusions revealed several developmental events including nucleocapsid assembly, envelopment of nucleocapsids at the inner nuclear membrane and their enclosure by a thin vacuolar membrane. While contained within cytoplasmic vacuoles, enveloped virions acquired surface spikes. Cytoplasmic vacuoles containing virions subsequently coalesced and discharged mature virions at the cell surface into the lumen of the salivary gland duct. The data indicate that the ultrastructural development of GPCMV in the guinea pig salivary gland shows many similarities to that of human cytomegalovirus in humans. The salivary gland may provide a primary locus for virus shedding and horizontal transmission of cytomegalovirus.