Endogenous ecotropic mouse type C viruses deficient in replication and production of XC plaques.

Endogenous ecotropic type C viruses were induced by iodedeoxyuridine from nontransformed and chemically or spontaneously transformed clones of the C3H/10T1/2 cell line. Viruses produced by cells of certain transformed clones were N-tropic and formed large XC plaques. In contrast, viruses produced by nontransformed C3H/10T1/2 cells were not detectable in the XC plaque test. These XC- viruses infected mouse cells with high efficiency, as shown by the induction of murine leukemia virus group-specific antigens in infected cells, but virus production, as determined by DNA polymerase-containing particles, was extremely low. Upon growth in certain mouse cells these replication-deficient, XC(-) viruses converted to type C viruses that were similar in XC assays to N-tropic AKR virus (XC+).     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Susceptibility of influenza viruses to interferon and to poly(I) . Poly(C) determined by the plaque reduction method

Susceptibility of eight strains of influenza A and B viruses to interferon and to poly(I) . poly(C) were determined by the plaque reduction method. All strains tested were slightly less susceptible than vesicular stomatitis virus (VSV) in an established line of canine kidney (MDCK) cells. The 50% plaque depression doses (PD50) of poly(I) . poly(C) for influenza A and B viruses were as high as 3.0- to 4.5-fold and 6- to 18-fold that for VSV, respectively. The amounts of interferon required to inhibit plaque formation of influenza A and B viruses by 50% were 3.0-6.2 and 7.3-15.2 units/ml, respectively. The ratio of PD50 of poly(I) . poly(C) for each strain of influenza viruses tested to that for VSV in chick embryo cells was almost the same as in MDCK cells. Furthermore, in chick embryo cells, the strains of influenza virus tested were demonstrated to be much more susceptible to poly(I) . poly(C) than both Newcastle disease virus and vaccinia virus. It is suggested that influenza viruses may be relatively susceptible to interferon and to poly(I) . poly(C).     

Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus.

When influenza (H3N2) viruses from infected individuals are grown in embryonated chicken eggs, viruses are isolated which differ antigenically and structurally from viruses grown in mammalian Madin-Darby canine kidney (MDCK) cell culture [G.C. Schild, J.S. Oxford, J.C. de Jong, and R.G. Webster, Nature (London) 303:706-709, 1983]. To determine which of these viruses is most representative of virus replicating in the infected individual, a region of the HA gene of virus present in original clinical samples was amplified by using the polymerase chain reaction and sequenced directly. Comparison of 170 amino acid residues of HA1 flanking and containing the receptor-binding site and antigenic sites indicated that over this region, the HA of virus replicating in the infected individual was identical to that of virus after growth in MDCK cells and was distinct from the HA of viruses grown in eggs. Therefore, cultivation of human influenza H3N2 virus in mammalian MDCK cells results in a virus similar to the predominant population of virus found in the infected individual.     

Genetic recombination for antigenic markers of antigenically different strains of influenza B virus

Incorporation of trypsin in agar overlay or fluid maintenance media resulted in enhancement of plaquing efficiency and replication of influenza B viruses in primary chicken embryo fibroblasts. Using this improved technique, recombination was attempted with two serologically distinct strains of influenza B virus, B/Lee/40 and B/Massachusetts/1/71. After mixed infection, two virus clones were selected and characterized in detail. Hemagglutination inhibition and neuraminidase inhibition tests showed that these viruses are reciprocal antigenic recombinants with hemagglutinin derived from one parent and neuraminidase from the other. Serological examinations of the antisera to these recombinants confirmed the results. The frequency of recombination was high in the present system and 64% of the virus clones isolated without selection from the mixed yield were recombinants. This high recombination frequency is consistent with the genomic reassortment that is characteristic of recombination of influenza A viruses.     

Development of a new cell system for the infectivity assay of dengue viruses: plaque formation and virus growth of prototype and wild-type dengue virus strains in a newly established cell line, GK

A newly established cell line, GK, derived from the kidney tissue of Mongolian gerbils, produced plaques by infection of prototype and wild-type dengue virus strains. Both prototype and wild strains of type 2 virus grew in GK cells and formed plaques at 35.5 C and at 31 C, while types 1, 3, and 4 wild strains grew and formed plaques only at 31 C. In GK cells, plaque formation and the growth of dengue viruses depended on the high (35.5 C) and low (31 C) incubation temperatures. Virus yields in GK cells of all the 14 dengue virus strains tested, including four prototype and ten wild-type viruses, were 5 to 1,000-times lower than those in C6/36 cells. After five serial passages in GK cells, types 2, 3, and 4 prototype viruses and type 2 wild strain increased virus yields, and one strain of prototype virus and one strain of wild-type virus decreased mouse neurovirulence.     

2015-2020年大连市流感病毒分离鉴定情况分析

对2015-2020年大连市流感病毒分离鉴定情况进行对比分析, 为大连市流感防控工作提供参考。

采集大连市2家国家级流感监测哨点医院的流感样病例咽拭子样本, 用MDCK细胞和鸡胚分别进行病毒分离培养, 并采用HA试验和HI试验对分离的病毒滴度和型别进行鉴定。

2015-2020年共分离培养流感病毒核酸检测阳性的咽拭子1 055份, 其中MDCK细胞分离出流感病毒501株, 鸡胚分离出流感病毒72株, 总体病毒分离率54.31%。MDCK细胞分离出A(H1N1)、A(H3N2)、B(Victoria)和B(Yamagata)型病毒, 鸡胚对A(H3N2)型病毒不敏感, 但可以分离出A(H1N1)、B(Victoria)和B(Yamagata)型病毒。每年的优势毒株虽不同, 但分离流感病毒的月份均在流行季内, 与北方流行形势一致。

MDCK细胞与鸡胚的流感病毒分离率不同。大连市每年流感流行的优势株和流行程度虽不同, 但流行程度处于相对平稳状态。

     

Host-Specific Hemagglutination of Influenza A (H1N1) Virus

H1N1 strains of influenza A virus isolated during the influenza season of 1991–92 were divided into two groups according to the property of host-specific hemagglutination. Group 1 viruses agglutinated human and chicken red blood cells. Group 2 viruses agglutinated human but not chicken red blood cells. The viruses of both groups, however, showed the same antigenic structure determined with ferret antisera. The virus clones which were plaque-purified twice from a group 2 virus retained the characteristic of host-specific hemagglutination after five successive passages in MDCK cells, indicating that this phenomenon is genetically determined. However, the amino acid, sequences of the hemagglutinin (HA) polypeptides deduced from the nucleotide sequences of the HA gene of the two groups did not show any differences between them. This suggests a difference in amino acids in some other polypeptide(s), which affects the host-specific hemagglutination.     

Phenotypic and Genetic Characterization of Avian Influenza H5N2 Viruses with Intra- and Inter-Duck Variations in Taiwan

Background

Human infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past.

Methods

Two duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved.

Results

The DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells.

Conclusion

These results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission.     

Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes differ in their ability to support assembly

The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.     

Specific biochemical features of replication of clinical influenza viruses in human intestinal cell culture

Influenza A viruses isolated from the respiratory tract of patients with influenza were cultured in human intestinal epithelium cells (CACO-2 line). The CACO-2 cells were found to be 100-fold more susceptible to the clinical viruses than MDCK cells and chicken embryos. On passaging in CACO-2 cells, clinical isolates of the subtype H3N2 retained the original "human" phenotype and agglutinated human but not chicken erythrocytes, whereas on passaging in MDCK cells the virus phenotype changed to the "avian" one. On comparison with laboratory strains (grown in chicken embryos or MDCK cells), the clinical viruses were characterized by higher stability of the anti-interferon protein NS1 but had a reduced synthesis of the matrix protein M1, and this could facilitate the virus adaptation and escape of the infected cells from immune attack in the human body. The increased tropism to the human CACO-2 cells correlated with higher adsorption of the clinical viruses on cellular receptors. However, in the CACO-2 and MDCK cells the ratio of sialyl-containing glycoreceptors of the 2-3 and 2-6 type was similar. These observations indicated that not only sialic acid residues were involved in the adsorption and penetration of the clinical viruses into human cells, but also the protein moiety of the cellular receptor itself and/or an additional cellular coreceptor. Thus, clinical influenza viruses are shown to possess a specific mechanism of sorption and entry into human epithelial cells, which is responsible for their higher tropism to human cells and is unlike such a mechanism in canine cells.     

Differences in pathogenicity among cloned sublines of a murine leukemia virus.

Five clones of the lymphatic leukemia virus 334C were isolated by a procedure designed to maintain homogeneity of the clones. Three of these induced leukemia in mice with the time course of the uncloned parental virus, one induced leukemia with a delayed time course, and one seemed to be biologically inactive. When the clone inducing leukemia most rapidly and the clone inducing leukemia least rapidly were subcloned, the subclones retained the leukemogenicity of the parental clones. The electrophoretic patterns of purified virion proteins and hybridization of viral RNAs with virus-specific DNA suggest that these clones are two closely related variants, not unrelated viruses. Furthermore, in mice infected with these two clones, viral RNA appears in thymuses and spleens at the same time after infection and at nearly the same concentrations. Thus, variations in leukemogenicity can be determined by a genetic property of an ecotropic leukemia virus, and this property is expressed in some manner more subtle than simple control of replication.     

Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.

In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types.     

Antigenic and phenotypic variants of a virulent avian influenza virus selected during replication in ducks

To evaluate the replication of a highly virulent avian influenza A virus in a potential reservoir host, mallard ducks (Anas platyrhynchos) were inoculated with the virulent strain A/Ty/Ont/7732/66 (H5N9). Viruses recovered from the ducks were analyzed by hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) and found to possess antigenically altered viral hemagglutinins. Plaque formation on the Madin-Darby Canine Kidney (MDCK) cell line and on primary chicken embryo cells was investigated, and isolates recovered from the ducks differed from the wild type by being unable to form plaques on MDCK cells without trypsin. This phenotype did not appear to be due to inefficient cleavage of the hemagglutinin by host cell proteases since hemagglutinin immunoprecipitated from cell lysates was cleaved. Although the plaquing phenotype suggested attenuation of the isolates from the ducks, they were not significantly altered in their virulence for chickens shown by infectivity studies in vivo. These results indicate that replication of influenza A/Ty/Ont/7732/66 virus in ducks can produce antigenic and phenotypic variants which are still highly virulent for domestic poultry.     

Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures.

Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.     

Selective inhibition of translation of the mRNA coding for measles virus membrane protein at elevated temperatures.

The elevation of culture temperatures of C6 cells that were persistently infected with the Lec strain of the subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) resulted in immediate selective inhibition of membrane (M) protein synthesis. This phenomenon was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cytoplasmic lysates and immunoprecipitation with monoclonal antibody against the M protein in short-time labeling experiments. The synthesis of various viral mRNAs in the presence of actinomycin D decreased gradually at similar rates after a shift to 39 degrees C. No specific disappearance of the mRNA coding for the M protein was observed when viral RNAs isolated from the infected cells were compared before and after a shift up by Northern blot analysis. Results of pulse-chase experiments did not show any significant difference in M protein stability between 35 and 39 degrees C. This rapid block of M protein synthesis was observed not only in Vero cells that were lytically infected with plaque-purified clones from the Lec strain, clones isolated from C6/SSPE cells and the standard Edmonston strain of measles virus but also in CV1, MA160, and HeLa cells that were lytically infected with the Edmonston strain. Poly(A)+ RNAs that were extracted from C6/SSPE cells before and after a shift to 39 degrees C produced detectable phospho, nucleocapsid, and M proteins in cell-free translation systems at 32 degrees C. Even higher incubation temperatures did not demonstrate the selective depression of M protein synthesis described above in vitro. All these data indicate that M protein synthesis of measles virus is selectively suppressed at elevated temperatures because of an inability of the translation apparatus to interact with the M protein-encoded mRNA.