Ovine Fetal and Placental Lesions and Cellular Tropism in Natural Rift Valley Fever Virus Infections.

Infection with Rift Valley fever phlebovirus (RVFV) causes abortion storms and a wide variety of outcomes for both ewes and fetuses. Sheep fetuses and placenta specimens were examined during the 2010-2011 River Valley fever (RVF) outbreak in South Africa. A total of 72 fetuses were studied of which 58 were confirmed positive for RVF. Placenta specimens were available for 35 cases. Macroscopic lesions in fetuses were nonspecific and included marked edema and occasional hemorrhages in visceral organs. Microscopically, multifocal hepatic necrosis was present in 48 of 58 cases, and apoptotic bodies, foci of liquefactive hepatic necrosis (primary foci), and eosinophilic intranuclear inclusions in hepatocytes were useful diagnostic features. Lymphocytolysis was present in all lymphoid organs examined with the exception of thymus and Peyer's patches, and pyknosis or karyorrhexis was often present in renal glomeruli. The most significant histologic lesion in the placenta was necrosis of trophoblasts and endothelial cells in the cotyledonary and intercotyledonary chorioallantois. Immunolabeling for RVFV was most consistent in trophoblasts of the cotyledon or caruncle. Other antigen-positive cells included hepatocytes, renal tubular epithelial, juxtaglomerular and extraglomerular mesangial cells, vascular smooth muscle, endothelial and adrenocortical cells, cardiomyocytes, Purkinje fibers, and macrophages. Fetal organ samples for diagnosis must minimally include liver, kidney, and spleen. From the placenta, the minimum recommended specimens for histopathology include the cotyledonary units and caruncles from the endometrium, if available. The diagnostic investigation of abortion in endemic areas should always include routine testing for RVFV, and a diagnosis during interepidemic periods might be missed if only limited specimens are available for examination.

Lesions and Cellular Tropism of Natural Rift Valley Fever Virus Infection in Young Lambs.

A clear distinction can be made regarding the susceptibility to and the severity of lesions in young lambs when compared to adult sheep. In particular, there are important differences in the lesions and tropism of Rift Valley fever virus (RVFV) in the liver, kidneys, and lymphoid tissues of young lambs. A total of 84 lambs (<6 weeks old), necropsied during the 2010 to 2011 Rift Valley fever (RVF) outbreak in South Africa, were examined by histopathology and immunohistochemistry (IHC). Of the 84 lambs, 71 were positive for RVFV. The most striking diagnostic feature in infected lambs was diffuse necrotizing hepatitis with multifocal liquefactive hepatic necrosis (primary foci) against a background of diffuse hepatocellular death. Lymphocytolysis was present in all lymphoid organs except for the thymus. Lesions in the kidney rarely progressed beyond hydropic change and occasional pyknosis or karyolysis in renal tubular epithelial cells. Viral antigen was diffusely present in the cytoplasm of hepatocytes, but this labeling was noticeably sparse in primary foci. Immunolabeling for RVFV in young lambs was also detected in macrophages, vascular smooth muscle cells, adrenocortical epithelial cells, renal tubular epithelial cells, renal perimacular cells, and cardiomyocytes. RVFV immunolabeling was also often present in capillaries and small blood vessels either as non-cell-associated viral antigen, as antigen in endothelial cells, or intravascular cellular debris. Specimens from the liver, spleen, kidney, and lungs were adequate to confirm a diagnosis of RVF. Characteristic lesions were present in these organs with the liver and spleen being the most consistently positive for RVFV by IHC.

Lesions and Cellular Tropism of Natural Rift Valley Fever Virus Infection in Adult Sheep.

Rift Valley fever (RVF) is a mosquito-borne disease that affects both ruminants and humans, with epidemics occurring more frequently in recent years in Africa and the Middle East, probably as a result of climate change and intensified livestock trade. Sheep necropsied during the 2010 RVF outbreak in South Africa were examined by histopathology and immunohistochemistry (IHC). A total of 124 sheep were available for study, of which 99 cases were positive for RVF. Multifocal-random, necrotizing hepatitis was confirmed as the most distinctive lesion of RVF cases in adult sheep. Of cases where liver, spleen, and kidney tissues were available, 45 of 70 had foci of acute renal tubular epithelial injury in addition to necrosis in both the liver and spleen. In some cases, acute renal injury was the most significant RVF lesion. Immunolabeling for RVFV was most consistent and unequivocal in liver, followed by spleen, kidney, lung, and skin. RVFV antigen-positive cells included hepatocytes, adrenocortical epithelial cells, renal tubular epithelial cells, macrophages, neutrophils, epidermal keratinocytes, microvascular endothelial cells, and vascular smooth muscle. The minimum set of specimens to be submitted for histopathology and IHC to confirm or exclude a diagnosis of RVFV are liver, spleen, and kidney. Skin from areas with visible crusts and lung could be useful additional samples. In endemic areas, cases of acute renal tubular injury should be investigated further if other more common causes of renal lesions have already been excluded. RVFV can also cause an acute infection in the testis, which requires further investigation.

Lack of Marburg Virus Transmission From Experimentally Infected to Susceptible In-Contact Egyptian Fruit Bats.

Egyptian fruit bats (Rousettus aegyptiacus) were inoculated subcutaneously (n = 22) with Marburg virus (MARV). No deaths, overt signs of morbidity, or gross lesions was identified, but microscopic pathological changes were seen in the liver of infected bats. The virus was detected in 15 different tissues and plasma but only sporadically in mucosal swab samples, urine, and fecal samples. Neither seroconversion nor viremia could be demonstrated in any of the in-contact susceptible bats (n = 14) up to 42 days after exposure to infected bats. In bats rechallenged (n = 4) on day 48 after infection, there was no viremia, and the virus could not be isolated from any of the tissues tested. This study confirmed that infection profiles are consistent with MARV replication in a reservoir host but failed to demonstrate MARV transmission through direct physical contact or indirectly via air. Bats develop strong protective immunity after infection with MARV.

Experimental Infection of Domestic Pigs (Sus scrofa) with Rift Valley Fever Virus.

Rift valley fever (RVF), caused by the RVF virus (RVFV), is a vector-borne zoonotic disease that primarily affects domestic ruminants. Abortion storms and neonatal deaths characterise the disease in animals. Humans develop flu-like symptoms, which can progress to severe disease. The susceptibility of domestic pigs (Sus scrofa domesticus) to RVFV remains unresolved due to conflicting experimental infection results. To address this, we infected two groups of pregnant sows, neonates and weaners, each with a different RVFV isolate, and a third group of weaners with a mixture of the two viruses. Serum, blood and oral, nasal and rectal swabs were collected periodically, and two neonates and a weaner from group 1 and 2 euthanised from 2 days post infection (DPI), with necropsy and histopathology specimens collected. Sera and organ pools, blood and oronasorectal swabs were tested for RVFV antibodies and RNA. Results confirmed that pigs can be experimentally infected with RVFV, although subclinically, and that pregnant sows can abort following infection. Presence of viral RNA in oronasorectal swab pools on 28 DPI suggest that pigs may shed RVFV for at least one month. It is concluded that precautions should be applied when handling pig body fluids and carcasses during RVF outbreaks.

Diagnostic sensitivity and specificity of immunohistochemistry for the detection of rabies virus in domestic and wild animals in South Africa.

We estimated the diagnostic sensitivity (DSe) and specificity (DSp) of an immunohistochemistry (IHC) protocol compared to the direct fluorescent antibody test (DFAT), which is the gold standard test for rabies diagnosis. We obtained brain samples from 199 domestic and wild animal cases (100 DFAT-negative, 99 DFAT-positive), by convenience sampling from 2 government-accredited rabies virus (RABV) testing laboratories in South Africa, between February 2015 and August 2017. Tissues that had been stored at 4-8°C for several days to weeks at the 2 accredited laboratories were formalin-fixed and paraffin-embedded. Nighty-eight cases tested IHC-positive using a polyclonal anti-RABV nucleoprotein antibody and a polymer detection system. The overall DSe and DSp for the RABV IHC test were 98% (95% CI: 93-100%) and 99% (95% CI: 95-100%), respectively. Domestic dogs accounted for 41 of 98 RABV IHC-positive cases, with the remainder in 4 domestic cats, 25 livestock, and 28 wildlife. Herpestidae species, including 7 meerkats and 9 other mongoose species, were the most frequently infected wild carnivores, followed by 11 jackals. Three cases in domestic dogs had discordant test results; 2 cases were IHC-/DFAT+ and 1 case was IHC+/DFAT-. Considering the implications of a false-negative rabies diagnosis, participating in regular inter-laboratory comparisons is vital, and a secondary or confirmatory method, such as IHC, should be performed on all submitted specimens, particularly negative cases with human contact history.

Insights into the Pathogenesis of Viral Haemorrhagic Fever Based on Virus Tropism and Tissue Lesions of Natural Rift Valley Fever.

Rift Valley fever phlebovirus (RVFV) infects humans and a wide range of ungulates and historically has caused devastating epidemics in Africa and the Arabian Peninsula. Lesions of naturally infected cases of Rift Valley fever (RVF) have only been described in detail in sheep with a few reports concerning cattle and humans. The most frequently observed lesion in both ruminants and humans is randomly distributed necrosis, particularly in the liver. Lesions supportive of vascular endothelial injury are also present and include mild hydropericardium, hydrothorax and ascites; marked pulmonary congestion and oedema; lymph node congestion and oedema; and haemorrhages in many tissues. Although a complete understanding of RVF pathogenesis is still lacking, antigen-presenting cells in the skin are likely the early targets of the virus. Following suppression of type I IFN production and necrosis of dermal cells, RVFV spreads systemically, resulting in infection and necrosis of other cells in a variety of organs. Failure of both the innate and adaptive immune responses to control infection is exacerbated by apoptosis of lymphocytes. An excessive pro-inflammatory cytokine and chemokine response leads to microcirculatory dysfunction. Additionally, impairment of the coagulation system results in widespread haemorrhages. Fatal outcomes result from multiorgan failure, oedema in many organs (including the lungs and brain), hypotension, and circulatory shock. Here, we summarize current understanding of RVF cellular tropism as informed by lesions caused by natural infections. We specifically examine how extant knowledge informs current understanding regarding pathogenesis of the haemorrhagic fever form of RVF, identifying opportunities for future research.

Vaccination with Rift Valley fever virus live attenuated vaccine strain Smithburn caused meningoencephalitis in alpacas.

Rift Valley fever (RVF) is a zoonotic, viral, mosquito-borne disease that causes considerable morbidity and mortality in humans and livestock in Africa and the Arabian Peninsula. In June 2018, 4 alpaca inoculated subcutaneously with live attenuated RVF virus (RVFV) Smithburn strain exhibited pyrexia, aberrant vocalization, anorexia, neurologic signs, and respiratory distress. One animal died the evening of inoculation, and 2 at ~20 d post-inoculation. Concern regarding potential vaccine strain reversion to wild-type RVFV or vaccine-induced disease prompted autopsy of the latter two. Macroscopically, both alpacas had severe pulmonary edema and congestion, myocardial hemorrhages, and cyanotic mucous membranes. Histologically, they had cerebral nonsuppurative encephalomyelitis with perivascular cuffing, multifocal neuronal necrosis, gliosis, and meningitis. Lesions were more severe in the 4-mo-old cria. RVFV antigen and RNA were present in neuronal cytoplasm, by immunohistochemistry and in situ hybridization (ISH) respectively, and cerebrum was also RVFV positive by RT-rtPCR. The virus clustered in lineage K (100% sequence identity), with close association to Smithburn sequences published previously (identity: 99.1-100%). There was neither evidence of an aberrant immune-mediated reaction nor reassortment with wild-type virus. The evidence points to a pure infection with Smithburn vaccine strain as the cause of the animals' disease.

Retrospective phylogenetic analyses of formalin-fixed paraffin-embedded samples from the 2011 Rift Valley fever outbreak in South Africa, through sequencing of targeted regions.

The last major Rift Valley fever outbreak in South Africa was between 2008 and 2011. Viruses isolated between 2008 and 2010 were phylogenetically assigned to Lineage C, Lineage K and the novel lineage H. The 2011 outbreaks occurred primarily in the Eastern, Western and Northern Cape provinces, with no sequence data or phylogenetic relationship published. Samples from these outbreaks were submitted to the Faculty of Veterinary Sciences, University of Pretoria, for immunohistochemical confirmation of Rift Valley fever phlebovirus presence. These samples were formalin-fixed and paraffin-embedded (FFPE) and stored at the Pathology section for several years. This study describes a modified extraction method used to obtain RNA from the FFPE samples, as well as the primer combinations used to phylogenetically classify them as belonging to the novel lineage H.

Rift Valley Fever Viral RNA Detection by In Situ Hybridization in Formalin-Fixed, Paraffin-Embedded Tissues.

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.

Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice.

The NSs protein encoded by the S segment of Rift Valley fever virus (RVFV) is the major virulence factor, counteracting the host innate antiviral defence. It contains five highly conserved cysteine residues at positions 39, 40, 149, 178 and 194, which are thought to stabilize the tertiary and quaternary structure of the protein. Here, we report significant differences between clinical, virological, histopathological and host gene responses in BALB/c mice infected with wild-type RVFV (wtRVFV) or a genetic mutant having a double cysteine-to-serine substitution at residues 39 and 40 of the NSs protein (RVFV-C39S/C40S). Mice infected with the wtRVFV developed a fatal acute disease; characterized by high levels of viral replication, severe hepatocellular necrosis, and massive up-regulation of transcription of genes encoding type I and -II interferons (IFN) as well as pro-apoptotic and pro-inflammatory cytokines. The RVFV-C39S/C40S mutant did not cause clinical disease and its attenuated virulence was consistent with virological, histopathological and host gene expression findings in BALB/c mice. Clinical signs in mice infected with viruses containing cysteine-to-serine substitutions at positions 178 or 194 were similar to those occurring in mice infected with the wtRVFV, while a mutant containing a substitution at position 149 caused mild, non-fatal disease in mice. As mutant RVFV-C39S/C40S showed an attenuated phenotype in mice, the molecular mechanisms behind this attenuation were further investigated. The results show that two mechanisms are responsible for the attenuation; (1) loss of the IFN antagonistic propriety characteristic of the wtRVFV NSs and (2) the inability of the attenuated mutant to degrade Proteine Kinase R (PKR).

Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep.

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.