The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging

The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.     

Replication of dengue-2 virus in cultured human lymphoblastoid cells and subpopulations of human peripheral leukocytes.

We studied the susceptibility of four human lymphoblastoid cell lines (HCL) and of subpopulations of circulating peripheral human leukocytes to dengue-2 virus infection. HCL with B cell characteristics (Raji, Wil 2WT, 8866), B-type peripheral lymphocytes, and macrophages were productively infected by dengue-2 virus. In contrast, an HCL with T cell characteristics (MOLT-4), T type peripheral lymphocytes, and polymorphonuclear (PMN) cells did not become infected and replicate dengue-2 virus. PMN cells did not adsorb dengue-2 virus, suggesting lack of viral receptors. However, T-type cultured lymphoblasts and T-type peripheral lymphocytes adsorbed dengue-2 virus, suggesting that the block in viral replication involves some stage of infection occurring after adsorption. Permissiveness of B-type HCL to dengue-2 virus infection was dependent on the virus seed used but the virus titers obtained among the susceptible HCL varied. HCL infected persistently with dengue-2 virus have been established. Human peripheral lymphocytes inoculated after cultivation for 3 days in complete medium alone or complete medium supplemented with mitogens replicated dengue-2 virus. In contrast, unstimulated peripheral lymphocytes inoculated immediately after isolation adsorbed dengue-2 but did not support its replication. Mitogen-treated and untreated macrophages replicated dengue-2 virus equally well. The efficiency of dengue-2 virus replication by macrophages was higher than that of peripheral lymphocytes but lower than that of HCL.     

甲/福流/8/58(H2N2)血凝素基因对该病毒在地鼠鼻甲中繁殖性状的影响

用在地鼠鼻甲繁殖不好、但在肺中繁殖较好的甲/福流/8/58(H2N2)流感病毒,与在地鼠的上下呼吸道均能较好地繁殖的H3N2型病毒重组,所获重组株(福R3),除HA基因是来自H2N2外,其它基因均来自H3N2。该重组病毒像亲本株H2N2株一样,在地鼠鼻甲繁不好。结果表明,编码甲/福流/8/58病毒的H2血凝素的基因影响着地鼠的组织嗜性。     

Isolation of a monoclonal antibody which blocks vaccinia virus infection.

We have isolated a monoclonal antibody, B2, that neutralizes vaccinia virus infection. B2 reacts with a trypsin-sensitive cell surface epitope. B2 does not neutralize infection of herpes simplex virus, suggesting that the B2-reactive epitope is specifically involved in vaccinia virus entry. A survey of 12 different cell lines reveals a correlation between B2 reactivity and susceptibility to vaccinia virus infection. In addition, B2 interferes with vaccinia virus adsorption to target cells. Taken together, the B2-reactive epitope is part of a receptor that appears important for vaccinia virus entry.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

甲3型流感病毒M2基因在痘苗病毒天坛株中的表达

为获得表达甲3型流感病毒(H3N2)M2蛋白的重组天坛株痘苗病毒RVJ1175M2,使用PCR方法扩增流感病毒全长M2基因,将其克隆到天坛株痘苗病毒同源重组质粒pJSC1175中,获得重组质粒pJSC1175M2,通过与痘苗病毒载体同源重组,构建了含流感病毒M2基因的重组痘苗病毒株RVJ1175M2。PCR检测结果证明,流感病毒(H3N2)M2蛋白基因准确插入到天坛株痘苗病毒TK区;Western blot、免疫荧光和流式细胞计数表明重组病毒RVJ1175M2可以有效地表达M2蛋白,表达的M2蛋白有两条带,分别为15kD和13kD,与相关文献报道一致;M2蛋白可有效分布在感染细胞的细胞膜上。这些结果表明重组痘苗病毒株RVJ1175M2可以有效地表达流感病毒M2蛋白,为使用表达M2蛋白的不同类型疫苗进行广谱流感疫苗效果的比较研究奠定了基础。     

Properties of Noninfectious and Transforming Viruses Released by Murine Sarcoma Virus-Induced Hamster Tumor Cells

The cell culture lines HTG2 and HTG3 were established from a transplantable hamster tumor induced by a murine sarcoma virus (strain Gz-MSV) after 17 and 60 in vivo passages, respectively. The viruses released by these two cell lines markedly differ in morphology, antigenic composition, infectivity, transforming ability, and enzymatic activity. HTG2 virions contained the sarcoma genome but were noninfectious for mouse and hamster cells (S+H-virus). HTG3 virions transformed hamster but not mouse cells. Whereas HTG2 cells and its virus contained murine type C virus gs-1 antigen, all HTG3 clonal lines expressed both murine and hamster type C virus gs-1 antigens. The RNA-dependent DNA polymerase activity of HTG2 virus was very low, whereas that of HTG3 virus was relatively high. HTG2 virions contained electron-lucent centers only. HTG3 virus consisted of the expected mixture of virions with electron-dense and electron-lucent centers. Many broken or incomplete virions were present in both viruses. HTG2 virus is a noninfectious "defective" sarcoma virus without detectable helper virus. Data obtained in these experiments suggest that HTG3 virus is a hamster type C virus pseudotype of Gz-MSV (Gz-MSV [HaLV]). The genome of Gz-MSV is capable of antigenic expression in heterologous cells and in the presence of heterologous viruses. Attempts to chemically activate hamster type C virus (HaLV) from HTG2 cells were unsuccessful. The HTG1 cell culture line, established from another Gz-MSV-induced hamster tumor, initially released a virus indistinguishable from the HTG2 virus. After in vitro passage, spontaneous activation of HaLV occurred in HTG1 cells, and the resultant Gz-MSV (HaLV) had properties similar to those of the HTG3 virus.     

The enantiomers of carbocyclic 5'-norguanosine: activity towards Epstein-Barr virus

(-)-5'-noraristeromycin (1) has shown antiviral activity towards, particularly cytomegalovirus, vaccinia virus and measles while its (+)-enantiomer (2) is effective towards hepatitis B virus. To determine if the antiviral characteristics of 1 and 2 extended to the guanine analogues (3 and 4), these enantiomers were prepared and evaluated against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), cytomegalovirus (CMV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus type 6 (HHV-6), human herpes virus type 8 (HHV-8), vaccinia virus (VV), cowpox virus (CV), vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), hepatitis B virus (HBV), and human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The only activity found for 3 was for Epstein-Barr virus in VCA Elisa (EC50 0.78 microg/mL), immunofluorescence assay for VCA or gp 350/250 (1.8-4.0 microg/mL) and DNA hybridization (EC50 0.82 microg/mL) assays with no accompanying toxicity seen in the host Daudi cells. No activity was noted for 4.     

Subtype cross-reactive, infection-enhancing antibody responses to influenza A viruses.

Antibody-dependent enhancement of the uptake of influenza A virus by Fc receptor-bearing cells was analyzed by using virus strains of the three human influenza A virus subtypes, A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and A/Port Chalmers/1/73 (H3N2). Immune sera obtained from mice following primary infection with an H1N1, H2N2, or H3N2 subtype virus neutralized only virus of the same subtype; however, immune sera augmented the uptake of virus across subtypes. Immune sera from H1N1-infected mice augmented uptake of the homologous (H1N1) and H2N2 viruses. Antisera to the H2N2 virus augmented the uptake of virus of all subtypes (H1N1, H2N2, or H3N2). Antisera to the H3N2 virus augmented the uptake of the homologous (H3N2) and H2N2 viruses. These results show that subtype cross-reactive, nonneutralizing antibodies augment the uptake of influenza A virus strains of different subtypes. Antibodies to neuraminidase may contribute to the enhanced uptake of viruses of a different subtype, because N2-specific monoclonal antibodies augmented the uptake of both A/Japan/305/57 (H2N2) and A/Port Chalmers/1/73 (H3N2) viruses.     

Influenza B virus BM2 protein is a crucial component for incorporation of viral ribonucleoprotein complex into virions during virus assembly

Influenza B virus contains four integral membrane proteins in its envelope. Of these, BM2 has recently been found to have ion channel activity and is considered to be a functional counterpart to influenza A virus M2, but the role of BM2 in the life cycle of influenza B virus remains unclear. In an effort to explore its function, a number of BM2 mutant viruses were generated by using a reverse genetics technique. The BM2DeltaATG mutant virus synthesized BM2 at markedly lower levels but exhibited similar growth to wild-type (wt) virus. In contrast, the BM2 knockout virus, which did not produce BM2, did not grow substantially but was able to grow normally when BM2 was supplemented in trans by host cells expressing BM2. These results indicate that BM2 is a required component for the production of infectious viruses. In the one-step growth cycle, the BM2 knockout virus produced progeny viruses lacking viral ribonucleoprotein complex (vRNP). The inhibited incorporation of vRNP was regained by trans-supplementation of BM2. An immunofluorescence study of virus-infected cells revealed that distribution of hemagglutinin, nucleoprotein, and matrix (M1) protein of the BM2 knockout virus at the apical membrane did not differ from that of wt virus, whereas the sucrose gradient flotation assay revealed that the membrane association of M1 was greatly affected in the absence of BM2, resulting in a decrease of vRNP in membrane fractions. These results strongly suggest that BM2 functions to capture the M1-vRNP complex at the virion budding site during virus assembly.     

Distinct domains of the influenza a virus M2 protein cytoplasmic tail mediate binding to the M1 protein and facilitate infectious virus production

The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates. The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.     

Genomic arrangement of an adenovirus-simian virus 40 hybrid virus, Ad2+ND4del.

Ad2+ND4del is an adenovirus type 2-simian virus 40 hybrid virus nondefective for growth in human cells. The virus was first observed when stocks of Ad2+ND4, a hybrid isolated from primary monkey kidney cells, were propagated in human cells. This paper describes the DNA sequence at two sites of DNA recombination, the site of the left adenovirus type 2-simian virus 40 junction and the site of a deletion of internal simian virus 40 sequences. Since the deletion was observed when the virus was switched from monkey to human cells, an analysis of gene expression in the region of DNA rearrangement may prove useful for the elucidation of molecular events that accompany virus growth in different hosts.     

Nonspecificity of the phenomenon of influenza virus phagocytosis by murine macrophages

Peritoneal macrophage cultures from intact mice and those immune to influenza virus A/PR/8/34 (HON1) were infected with homologous virus or influenza virus A/England/42/72 (H3N2) whereupon virus was isolated from chick embryos. It was established that in intact macrophages, both viruses duplicated similarly. Macrophages immune to virus HON1 equally disintegrated both in homologous virus and heterologous influenza virus H3N2.     

A replication-incompetent PB2-knockout influenza A virus vaccine vector

Vaccination is the primary form of protection from influenza virus infection. We recently developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that possesses a reporter gene (the green fluorescent protein gene) in the coding region of the PB2 segment. This virus replicated to high titers in PB2-expressing, but not unmodified, cells, suggesting its potential safety and feasibility as a vaccine. Here, we tested its efficacy in a murine model. The levels of IgG and IgA antibodies against influenza virus in sera, nasal washes, and bronchoalveolar lavage fluids of mice immunized with the PB2-KO virus were higher than those induced by a conventional inactivated vaccine. All PB2-KO virus-immunized mice survived challenges with lethal doses of influenza virus. Moreover, importantly, mice immunized with the PB2-KO virus produced antibodies against the reporter protein, suggesting that the PB2-KO virus has potential as a multivalent vaccine to combat infection with not only influenza virus but also other pathogens.     

Impact of Prior Seasonal H3N2 Influenza Vaccination or Infection on Protection and Transmission of Emerging Variants of Influenza A(H3N2)v Virus in Ferrets

Influenza H3N2 A viruses continue to circulate in swine and occasionally infect humans, resulting in outbreaks of variant influenza H3N2 [A(H3N2)v] virus. It has been previously demonstrated in ferrets that A(H3N2)v viruses transmit as efficiently as seasonal influenza viruses, raising concern over the pandemic potential of these viruses. However, A(H3N2)v viruses have not acquired the ability to transmit efficiently among humans, which may be due in part to existing cross-reactive immunity to A(H3N2)v viruses. Although current seasonal H3N2 and A(H3N2)v viruses are antigenically distinct from one another, historical H3N2 viruses have some antigenic similarity to A(H3N2)v viruses and previous exposure to these viruses may provide a measure of immune protection sufficient to dampen A(H3N2)v virus transmission. Here, we evaluated whether prior seasonal H3N2 influenza virus vaccination or infection affects virus replication and transmission of A(H3N2)v virus in the ferret animal model. We found that the seasonal trivalent inactivated influenza virus vaccine (TIV) or a monovalent vaccine prepared from an antigenically related 1992 seasonal influenza H3N2 (A/Beijing/32/1992) virus failed to substantially reduce A(H3N2)v (A/Indiana/08/2011) virus shedding and subsequent transmission to naive hosts. Conversely, ferrets primed by seasonal H3N2 virus infection displayed reduced A(H3N2)v virus shedding following challenge, which blunted transmission to naive ferrets. A higher level of specific IgG and IgA antibody titers detected among infected versus vaccinated ferrets was associated with the degree of protection offered by seasonal H3N2 virus infection. The data demonstrate in ferrets that the efficiency of A(H3N2)v transmission is disrupted by preexisting immunity induced by seasonal H3N2 virus infection.     

Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes.     

Molecular Genetic Study of the Interaction of Sindbis Virus E2 with Ross River Virus E1 for Virus Budding

Glycoprotein PE2 of Sindbis virus will form a heterodimer with glycoprotein E1 of Ross River virus that is cleaved to an E2/E1 heterodimer and transported to the cell plasma membrane, but this chimeric heterodimer fails to interact with Sindbis virus nucleocapsids, and very little budding to produce mature virus occurs upon infection with chimeric viruses. We have isolated in both Sindbis virus E2 and in Ross River virus E1 a series of suppressing mutations that adapt these two proteins to one another and allow increased levels of chimeric virus production. Two adaptive E1 changes in an ectodomain immediately adjacent to the membrane anchor and five adaptive E2 changes in a 12-residue ectodomain centered on Asp-242 have been identified. One change in Ross River virus E1 (Gln-411→Leu) and one change in Sindbis virus E2 (Asp-248→Tyr) were investigated in detail. Each change individually leads to about a 10-fold increase in virus production, and combined the two changes lead to a 100-fold increase in virus. During passage of a chimeric virus containing Ross River virus E1 and Sindbis virus E2, the E2 change was first selected, followed by the E1 change. Heterodimers containing these two adaptive mutations have a demonstrably increased degree of interaction with Sindbis virus nucleocapsids. In the parental chimera, no interaction between heterodimers and capsids was visible at the plasma membrane in electron microscopic studies, whereas alignment of nucleocapsids along the plasma membrane, indicating interaction of heterodimers with nucleocapsids, was readily seen in the adapted chimera. The significance of these findings in light of our current understanding of alphavirus budding is discussed.     

Virus-encoded b7-2 costimulation molecules enhance the protective capacity of a replication-defective herpes simplex virus type 2 vaccine in immunocompetent mice

Herpes simplex virus 2 (HSV-2) and, to a lesser extent, HSV-1 cause the majority of sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective HSV stimulates immune responses in animals but produces no progeny virus, making it potentially useful as a safe form of live vaccine against HSV. Because it does not replicate and spread in the host, however, replication-defective virus may have relatively limited capacity to solicit professional antigen presentation. We previously demonstrated that in mice devoid of B7-1 and B7-2 costimulation molecules, replication-defective HSV-2 encoding B7-1 or B7-2 induces stronger immune responses and protection against HSV-2 challenge than immunization with replication-defective virus alone. Here, we vaccinated wild-type mice fully competent to express endogenous B7 costimulation molecules with replication-defective HSV-2 or replication-defective virus encoding B7-2 and compared their capacities to protect against vaginal HSV-2 infection and disease. Replication-defective virus encoding B7-2 induced more IFN-γ-producing CD4 T cells than did replication-defective virus alone. Immunization with B7-2-expressing virus decreased challenge virus replication in the vaginal mucosa, genital and neurological disease, and mortality more effectively than did immunization with the parental replication-defective virus. Prior immunization with B7-expressing, replication-defective virus also effectively suppressed infection of the nervous system compared to immunization with the parental virus. Thus, B7 costimulation molecules expressed at the site of HSV infection can enhance vaccine efficacy even in a fully immunocompetent host.     

Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del(29-31) virus. We found that the M2-del(29-31) virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.     

Replication of Dengue Virus Type 2 in Aedes albopictus Cell Culture

The replication of type 2 dengue (D-2) virus in Aedes albopictus (Aal) mosquito cell cultures differed from that in vertebrate (LLC-MK2) rhesus monkey kidney cells. Virus readily replicated in Aal cells at either 30 or 37 C, but had no apparent effect on the host cell. Persistent infection was established with continual virus production for at least 6 months, although the virulence of progeny virus for both suckling mice and LLC-MK2 cells became attenuated. Density gradient analysis of infected Aal cell supernatant products indicated that only complete virus was released, in contrast to infected LLC-MK2 cells which also released incomplete virus. The surface antigens of the virus produced in Aal cells appeared to be considerably modified in that antiserum to vertebrate cell-produced D-2 virus did not block hemagglutination, whereas anti-Aal cell antiserum did. Virus infectivity could be neutralized by the antiserum to D-2 virus grown in vertebrate cells, however. Virus produced in LLC-MK2 cells did not demonstrate a similar host-cell modification. These results may reflect a difference in the mechanism by which D-2 virus matures in Aal cells.