Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.
Acid alpha naphthyl acetate esterase (ANAE) and combined ANAE-chloroacetate esterase cytochemistry was performed on 121 bone marrow aspirates from primary myelodysplastic syndromes (MDS) and a secondary dysplasia-megaloblastic anaemia (MA). The investigation demonstrated the presence of abnormal ANAE positive granulocyte populations in a significant proportion of cases. These cells, in which the staining patterns were characterized by atypical granular ANAE positivity and double ANAE-chloroacetate reactions, were shown immunologically to lack the receptor and antigenic characteristics of monocytes and morphologically to be granulocytes. Isoelectric focusing, however, indicated that the atypical esterase cytochemistry of these granulocytes was due to the presence of markedly increased concentrations of ANAE isoenzymes usually found in monocytes. Atypical ANAE-staining granulocytes were particularly evident in MDS marrows showing sideroblastic erythroid changes, whilst in MA they were mainly seen in cases of intermediate severity. It is suggested that these cells are associated with dysmyelopoietic changes in both malignant and non-malignant conditions.
Widespread use of a live-attenuated influenza vaccine (LAIV) in the United States (licensed as FluMist) raises the possibility that vaccine viruses will contribute gene segments to the type A influenza virus gene pool. Progeny viruses possessing new genotypes might arise from genetic reassortment between circulating wild-type (wt) and vaccine strains, but it will be difficult to predict whether they will be viable or exhibit novel properties. To begin addressing these uncertainties, reverse-genetics was used to generate 34 reassortant viruses derived from wt influenza virus A/Sydney/5/97 and the corresponding live vaccine strain. The reassortants contained different combinations of vaccine and wt PB2, PB1, PA, NP, M, and NS gene segments whereas all strains encoded wt HA and NA glycoproteins. The phenotypes of the reassortant strains were compared to wt and vaccine viruses by evaluating temperature-sensitive (ts) plaque formation and replication attenuation (att) in ferrets following intranasal inoculation. The results demonstrated that the vaccine virus PB1, PB2, and NP gene segments were dominant when introduced into the wt A/Sydney/5/97 genetic background, producing recombinant viruses that expressed the ts and att phenotypes. A dominant attenuated phenotype also was evident when reassortant strains contained the vaccine M or PA gene segments, even though these polypeptides are not temperature-sensitive. Although the vaccine M and NS gene segments typically are not associated with temperature sensitivity, a number of reassortants containing these vaccine gene segments did exhibit a more restricted ts phenotype. Overall, no reassortant strains were more virulent than wt, and in fact, 33 of the 34 recombinant viruses replicated less efficiently in infected ferrets. These results suggest that genetic reassortment between wt and vaccine strains is unlikely to produce viruses having novel properties that differ substantially from either progenitor, and that the likely outcome of reassortment will be attenuated viruses.
Summary. Acid alpha naphthyl acetate esterase (ANAE) and combined ANAE‐chloroacetate esterase cytochemistry was performed on 121 bone marrow aspirates from primary myelodysplastic syndromes (MDS) and a secondary dysplasia—megaloblastic anaemia (MA). The investigation demonstrated the presence of abnormal ANAE positive granulocyte populations in a significant proportion of cases. These cells, in which the staining patterns were characterized by atypical granular ANAE positivity and double ANAE‐chloroacetate reactions, were shown immunologically to lack the receptor and antigenic characteristics of monocytes and morphologically to be granulocytes. Isoelectric focusing, however, indicated that the atypical esterase cytochemistry of these granulocytes was due to the presence of markedly increased concentrations of ANAE isoenzymes usually found in monocytes. Atypical ANAE‐staining granulocytes were particularly evident in MDS marrows showing sideroblastic erythroid changes, whilst in MA they were mainly seen in cases of intermediate severity. It is suggested that these cells are associated with dysmyelopoietic changes in both malignant and non‐malignant conditions.
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