Performance of morphologic criteria for the diagnosis of cirrhosis in patients with non-alcoholic steatohepatitis compared to other etiologies of chronic liver disease: effect of level of training and experience.

PURPOSE: To compare the diagnostic performance of morphologic criteria for detection of cirrhosis in patients with alcoholic liver disease (ALD), hepatitis C (HCV), and non-alcoholic steatohepatitis (NASH). METHODS: One hundred patients (53 male) with different etiologies of chronic liver disease (NASH, n = 41; HCV, n = 39; and ALD, n = 20) and with different degrees of fibrosis on histopathologic examination (74 with cirrhosis) were retrospectively evaluated. Four readers (R1: fellowship-trained abdominal radiologist, R2: community attending radiologist, R3: senior radiology resident/research fellow, R4: junior radiology resident) analyzed the contrast-enhanced CTs for presence of commonly accepted morphologic changes of cirrhosis and portal hypertension. Each reader assigned an overall score (using a 5-point scale) for possibility of cirrhosis based on liver morphology and features of portal hypertension. Diagnostic performance, sensitivity, and specificity for the diagnosis of cirrhosis were calculated and compared between different etiologies of chronic liver disease. RESULTS: Performance of readers was affected by their level of training. Less experienced readers had overall lower sensitivity for diagnosis of cirrhosis in NASH group (R3: 81.5%, R4: 63.0% compared to 96.3% for both R1 and R2). Sensitivities for detection of NASH cirrhosis significantly decreased for less experienced readers in the absence of ascites (R3: 75.0%, R4: 62.0%) or other features of portal hypertension (R3: 50.0%; R4: 0%). The specificity was consistently high among different etiologies and for all readers (85.7-100%). Inter-reader agreement for morphologic features ranged widely from fair to almost perfect (k: 0.23-0.85). CONCLUSION: Cirrhotic changes in NASH are subtler and can be underestimated by less experienced readers.

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system.

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.

Enhanced green fluorescent protein expression may be used to monitor murine coronavirus spread in vitro and in the mouse central nervous system.

Targeted recombination was used to select mouse hepatitis virus isolates with stable and efficient expression of the gene encoding the enhanced green fluorescent protein (EGFP). The EGFP gene was inserted into the murine coronavirus genome in place of the nonessential gene 4. These viruses expressed the EGFP gene from an mRNA of slightly slower electrophoretic mobility than mRNA 4. EGFP protein was detected on a Western blot of infected cell lysates and EGFP activity (fluorescence) was visualized by microscopy in infected cells and in viral plaques. Expression of EGFP remained stable through at least six passages in tissue culture and during acute infection in the mouse central nervous system. These viruses replicated with similar kinetics and to similar final extents as wild-type virus both in tissue culture and in the mouse central nervous system (CNS). They caused encephalitis and demyelination in animals as wild-type virus; however, they were somewhat attenuated in virulence. Isogenic EGFP-expressing viruses that differ only in the spike gene and express either the spike gene of the highly neurovirulent MHV-4 strain or the more weakly neurovirulent MHV-A59 strain were compared; the difference in virulence and patterns of spread of viral antigen reflected the differences between parental viruses expressing each of these spike genes. Thus, EGFP-expressing viruses will be useful in the studies of murine coronavirus pathogenesis in mice.

The virulence of mouse hepatitis virus strain A59 is not dependent on efficient spike protein cleavage and cell-to-cell fusion.

The cleavage and fusion properties of recombinant murine hepatitis viruses (MHV) were examined to assess the role of the cleavage signal in determining the extent of S protein cleavage, and the correlation between cleavage and induction of cell-to-cell fusion. Targeted recombination was used to introduce amino acid substitutions into the cleavage signal of the fusion glycoprotein (spike or S protein) of MHV strain A59. The recombinants were then used to address the question of the importance of S protein cleavage and viral-mediated cell-to-cell fusion on pathogenicity. Our data indicate that cleavage of spike is not solely determined by the amino acid sequence at the cleavage site, but may also depend on sequences removed from the cleavage site. In addition, efficient cell-to-cell fusion is not necessary for virulence.