In Vitro Modification of Human Immunodeficiency Virus Type 1 (HIV-1) Infectivity by the U937 Cells

The effect of host cell factors on infectivity of human immunodeficiency virus type 1 (HIV-1) was studied by infecting a monoblastoid cell line (U937) or a T-cell line (MOLT-4) with a highly infective single clone of HIV-1 and comparing the infectivity of the produced viruses to different cell lines. Chronically infected U937 cells consistently produced viruses with minimal infectivity. This phenotypic change was host-dependent as the back-passage of the U937-produced low infective viruses into MOLT-4 cells resulted in regaining their original high infectivity. Southern and Northern blot analyses of the HIV-1 grown in U937 cells did not reveal any genomic difference between it and the virus grown it MOLT-4 cells. The radioimmunoprecipitation analysis of viral proteins showed that the HIV-1-infected U937 cells had a different pattern of envelope glycoproteins and core proteins, which well correlated with the low infectivity of the produced viruses. This experimental system using MOLT-4 and U937 cell lines would be useful to further explore host cell factor(s) which play an important role in the regulation of HIV-1 infectivity.     

清洁级实验动物四种病毒抗体玻片酶免疫检测试剂盒的研制

本文报道对清洁级实验动物应排除的四种病毒（淋巴细胞脉络丛脑膜炎病毒、小鼠脱脚病病毒、鼠肝炎病毒和仙台病毒）抗体玻片酶免疫（ＥＩＡ）检测试剂盒的研制。四种病毒感染的细胞和对照细胞经冷丙酮固定于载玻片上制成特异性抗原和对照抗原,此四种病毒的抗血清各１０份和ＳＰＦ小鼠血清２０份分别与四种病毒的特异性抗原和对照抗原进行ＥＩＡ交叉试验,结果显示,抗原只与其相应抗血清发生特异性显色反应,与非特异性小鼠血清和ＳＰＦ小鼠血清不显色。与ＨＩ或ＥＬＩＳＡ方法比较,通过对１１２份普通小鼠血清进行测试,结果表明,ＥＩＡ对仙台病毒抗体的检出率（１９．６％）显著高于（＜０．００５）ＨＩ（６．３％）,对小鼠脱脚病病毒抗体的检出率（２３．３％）与ＨＩ（２１．４％）无显著性差异（Ｐ＞０．０５）。ＥＩＡ对淋巴细胞脉络丛脑膜炎病毒和鼠肝炎病毒抗体的检出率分别为１．８％和７１．２％,ＥＬＩＳＡ对两种病毒抗体的检出率分别为１．８％和６７．６％,两种方法对两种病毒抗体的检出率无显著性差异（Ｐ＞０．０５）。重复性试验表明两批四种病毒抗体试剂盒对１０８份小鼠血清两次测定的符合率为９６～１００％。四种病毒的ＥＩＡ抗原在－１８℃保存１２个月或在２－８℃保存３     

Cellular proteinases trigger the infectivity of the influenza A and Sendai viruses.

It has been proposed that the pathogenicity of the influenza and Sendai virus is primarily determined by host cellular proteases that activate viral infectivity. We isolated trypsin-type serine proteases from rat lungs, candidates for the processing proteases of viral envelope glycoproteins, such as tryptase Clara localized in the Clara cells of the bronchial epithelium and mini-plasmin. These enzymes specifically cleave the precursor of fusion glycoprotein HA of influenza virus at Arg325, and the F0 of Sendai virus at Arg116 in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the induction of infectivity of these viruses. Proteolytic activation of viruses by these enzymes occurs extracellularly, probably on the surface and/or in the lumen of the respiratory tract. On the other hand, we isolated two compounds from human bronchial lavage, which inhibit the activity of tryptase Clara. One was a mucus protease inhibitor and the other was a pulmonary surfactant. These compounds inhibited multiple cycles of virus replication in vitro and in vivo, but did not themselves affect the hemagglutination and the infectivity of the virus. Administration of these compounds in the airway may be useful for preventing and treating infection with influenza virus and Sendai virus.     

Detection of serum antibodies using LBC cell-grown sialodacryoadenitis (SDA) virus.

The TG strain of sialodacryoadenitis (SDA) virus propagated in LBC cell culture (TGr/LBC) reacted strongly with anti-TGr rat serum in complement fixation (CF) tests, showing much higher titers with anti-TGr rat serum than mouse hepatitis virus MHV-NuU antigen. The antigenicity was not affected after ether treatment while infectivity was lost. The TGr/LBC antigen might be useful in seromonitoring for SDA infection in rats.     

Nucleopolyhedroviruses of forest and western tent caterpillars: cross-infectivity and evidence for activation of latent virus in high-density field populations

Abstract. 1. Cyclic population dynamics of forest caterpillars are often associated with epizootics of nucleopolyhedrovirus, but it is not known how these viruses persist between generations or through the fluctuations in host population density. 2. To explore the question of virus persistence at different phases of the population cycle, the nucleopolyhedroviruses of two species of tent caterpillar that co‐occur in British Columbia, Canada, Malacosoma californicum pluviale (western tent caterpillar) and Malacosoma disstria (forest tent caterpillar), were characterised. The cross‐infectivity of the viruses in these two host species was investigated to determine whether there might be a route for virus persistence via the alternative host species. Any virus produced in the cross‐infections was characterised to confirm true cross‐infection or to ascertain whether cross‐inoculation triggered latent virus persisting within the population. 3. The virus associated with forest tent caterpillars (MadiNPV) did not infect western tent caterpillars from low‐density populations, nor did it trigger a latent virus infection; however, inoculation of forest tent caterpillars from high‐density populations with virus from western tent caterpillars (McplNPV) resulted in viral infection, but without a dose–response relationship. 4. Analysis of DNA profiles of virus resulting from cross‐infection of the forest tent caterpillar with McplNPV, revealed that 88% of these infections were caused by MadiNPV rather than McplNPV; however the virus from all 44 infected individuals was identical and differed in DNA profile from the stock MadiNPV used for cross‐infection. This suggests strongly that forest tent caterpillars from high‐density field populations harbour a latent, persistent, or sublethal form of MadiNPV that was triggered by exposure to nucleopolyhedrovirus from the western tent caterpillar. 5. Virus was not activated in western tent caterpillars collected over 2 years of late population decline and the first year of population increase.     

Rhabdovirus-Based Vectors with Human Immunodeficiency Virus Type 1 (HIV-1) Envelopes Display HIV-1-Like Tropism and Target Human Dendritic Cells

We describe replication-competent, vaccine strain-based rabies viruses (RVs) that lack their own single glycoprotein and express, instead, a chimeric RV-human immunodeficiency virus type 1 (HIV-1) envelope protein composed of the ectodomain and transmembrane domains of HIV-1 gp160 and the cytoplasmic domain of RV G. The envelope proteins from both X4 (NL4-3)- and R5X4 (89.6)-tropic HIV-1 strains were utilized. These recombinant viruses very closely mimicked an HIV-1- like tropism, as indicated by blocking experiments. Infection was inhibited by SDF-1 on cells expressing CD4 and CXCR4 for both viruses, whereas RANTES abolished infection of cells expressing CCR5 in addition to CD4 in studies of the RV expressing HIV-1(89.6) Env. In addition, preincubation with soluble CD4 or monoclonal antibodies directed against HIV-1 gp160 blocked the infectivity of both G-deficient viruses but did not affect the G-containing RVs. Our results also indicated that the G-deficient viruses expressing HIV-1 envelope protein, in contrast to wild-type RV but similar to HIV-1, enter cells by a pH-independent pathway. As observed for HIV-1, the surrogate viruses were able to target human peripheral blood mononuclear cells, macrophages, and immature and mature human dendritic cells (DC). Moreover, G-containing RV-based vectors also infected mature human DC, indicating that infection of these cells is also supported by RV G. The ability of RV-based vectors to infect professional antigen-presenting cells efficiently further emphasizes the potential use of recombinant RVs as vaccines.     

Human lymphoblastoid cells as hosts for parvoviruses H-1 and rat virus.

A human T-cell line (Molt-4) was shown by viral hemagglutination and infectivity assays to support the replication of rat virus (RV) and H-1 virus. In addition, H-1 virus, but not RV, multiplied in two human B-cell lines, AV-1 and NC-37. The ability to bind radioactively labeled RV was demonstrated for each of the cell lines, but viral adsorption occurred to a greater degree with Molt-4 cells than with either AV-1 or NC-37 cells. After challenge with RV, virus-specific antigens were detected in cells of the B-cell lines by the indirect immunofluorescence technique. Infection of AV-1 or NC-37 cells by RV apparently results in an abortive cycle of virus replication. Differences among the three cell lines that might influence with H-1 virus or RV are discussed.     

The advantage of enzyme-linked immunosorbent assay (ELISA) as a method of microbiological monitoring for rat virus (RV).

An enzyme-linked immunosorbent assay (ELISA) was tested to detect antibodies against rat virus (RV). The purified ELISA antigens were prepared from rat embryonic cells infected with RV-13 (prototype strain) and UT-2 (Japanese isolate), respectively. Western blotting analysis confirmed that both of these antigens had three structural polypeptides (81 K, 61 K, and 59 K). Eleven laboratory and wild rat colonies in Japan were tested for rat virus contamination, serologically. No significant differences in the sero-positive ratio and the distributions of ELISA titers were demonstrated in the ELISA, using antigens from RV-13 and UT-2. ELISA was more sensitive and specific for detecting antibodies against RV from rat serum rather than hemagglutination inhibition (HI) test. This study also confirmed that the RV contaminated widely in colonies of laboratory and wild rats in Japan, and suggested that RV would have to be checked during the microbiological monitoring of laboratory rats.     

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity.     

Identification of trypsin I as a candidate for influenza A virus and Sendai virus envelope glycoprotein processing protease in rat brain

Extracellular cleavage of virus envelope fusion glycoprotein hemagglutinin (HA0) by host trypsin-like proteases is a prerequisite for the infectivity and pathogenicity of human influenza A viruses and Sendai virus. The common epidemic influenza A viruses are pneumotropic, but occasionally cause encephalopathy or encephalitis, although the HA0 processing enzyme in the brain has not been identified. In searching for the brain processing proteases, we identified a processing enzyme in rat brain that was inducible by infection with these viruses. The purified enzyme exhibited an apparent molecular mass of approximately 22 kDa on SDS-PAGE and the N-terminal amino acid sequence was consistent with that of rat pancreatic trypsin I. Its substrate specificities and inhibition profiles were the same as those of pancreatic trypsin I. In situ hybridization and immunohistochemical studies on trypsin I distribution revealed heavy deposits in the brain capillaries, particularly in the allocortex, as well as in clustered neuronal cells of the hippocampus. The purified enzyme efficiently processed the HA0 of human influenza A virus and the fusion glycoprotein precursor of Sendai virus. Our results suggest that trypsin I in the brain potentiates virus multiplication in the pathogenesis and progression of influenza-associated encephalopathy or encephalitis.     

In vitro recombinants of ground squirrel and woodchuck hepatitis viral DNAs produce infectious virus in squirrels.

Hepatitis B viruses of humans, woodchucks, ground squirrels, and ducks are similar biochemically but differ with respect to host range and pathogenicity. To pursue the genetic basis of these properties in the absence of a cell culture system for virus growth, we exploited the demonstrated infectivity of cloned hepatitis B virus DNA in whole animals. We constructed several recombinant molecules in vitro between cloned infectious genomes of woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV) and assayed the recombinants for infectivity after intrahepatic injection in ground squirrels, which support growth of GSHV but not WHV. Two of the recombinants molecules initiated productive infection; in one recombinant genome, 76% of the coding region for the major surface glycoprotein of GSHV and for the overlapping portion of the presumptive gene for DNA polymerase was replaced by WHV DNA; in the other, 29% of the same coding domain was replaced by WHV DNA. These findings demonstrate the feasibility of generating viable recombinants of hepatitis B viruses from different animal species and suggest that the major host range determinants are not encoded within the surface antigen gene of these viruses.     

Effect of kaolinite on the specific infectivity of reovirus

Abstract The infectivity of enteric viruses (e.g., poliovirus, rotavirus, reovirus) is prolonged when these viruses are adsorbed on naturally occurring particulates (sediments, clay minerals) in terrestrial and aquatic environments. Furthermore, in vitro assays of these and other particulate-associated viruses often display infectivity levels (specific infectivity) greater than those of the same concentration of viruses in the absence of particulates. This investigations attempted to identify interactions at the particulate-virus-cell interface and to define the mechanism(s) whereby the apparent infectivity of viruses is enhanced when complexed with particulates. Reovirus type 3 and the clay mineral, kaolinite, were used as the model systems. Scanning electron micrographs after critical point drying showed that kaolinite was not present on the surface of cell monolayers of L-929 mouse fibroblasts 3 h after inoculation with a kaolinite-reovirus complex. However, the virus was observed on the surface of the cells. No change in dispersion of the virus particles was observed nor was the integrity of the cell surface altered by kaolinite. These results indicated that kaolinite enhanced the transport of viral particles, in conjunction with diffusion and Brownian movement, to receptors for the reovirus on the cell surface.     

In situ cleavage of baculovirus occlusion-derived virus receptor binding protein P74 in the peroral infectivity complex

Proteolytic processing of viral membrane proteins is common among enveloped viruses and facilitates virus entry. The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) occlusion-derived virus (ODV) protein P74 is part of a complex of essential peroral infectivity factors (PIFs). Here we report that P74 is efficiently cleaved into two fragments of about equal size by an occlusion body (OB) endogenous alkaline protease during ODV release when AcMNPV OBs are derived from larvae. The cleavage is specific for P74, since the other known peroral infectivity factors in the same complex (PIF1, PIF2, and PIF3) were not cleaved under the same conditions. P74 cleavage was not observed in OBs produced in three different insect cell lines, suggesting a larval host origin of the responsible protease. P74 in OBs produced in larvae of two different host species was cleaved into fragments with the same apparent molecular mass, indicating that the virus incorporates a similar alkaline protease from different hosts. Coimmunoprecipitation analysis revealed that the two P74 subunit fragments remain associated with the recently discovered PIF complex. We propose that under in vivo ODV infection conditions, P74 undergoes two sequential cleavage events, the first one being performed by an ODV-associated host alkaline protease and the second carried out by trypsin in the host midgut.     

Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses: possible involvement in the pathogenesis of human H5N1 virus infection

Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.     

Purification of the Kilham Rat Virus

A purification procedure is described for the isolation of Kilham rat virus (RV) from infected suckling hamster kidney and liver suspensions. The procedure involved a combination of sonic treatment, differential centrifugation, butanol-chloroform extraction, agar column flow diffusion, and potassium tartrate density gradient centrifugation. The purified virus retained its infectivity and was specifically neutralized by RV hyperimmune antiserum. Electron micrographs from the RV band (density 1.31 g/ml) showed numerous homogeneous particles approximately 22 mmu in diameter.     

Identification of ectopic anionic trypsin I in rat lungs potentiating pneumotropic virus infectivity and increased enzyme level after virus infection.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. In search of such target processing proteases in the airway, we recently found a new candidate trypsin-like processing protease in rat lungs, which was induced by Sendai virus infection, and identified as ectopic rat anionic trypsin I. On SDS/PAGE under reducing and nonreducing conditions, the purified enzyme gave protein bands corresponding to 29 and 22 kDa, respectively, i.e. at the same positions as rat pancreatic anionic trypsin I. It exhibited an apparent molecular mass of 31 kDa on molecular sieve chromatography and its isoelectric point was pH 4.7. The amino-acid sequences of the N-terminus and proteolytic digest peptides of the purified enzyme were consistent with those of rat pancreatic anionic trypsin I. Its substrate specificities and inhibitor sensitivities were the same as those of the pancreatic enzyme. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and hemagglutinin of human influenza A virus, and potentiated the infectivity of Sendai virus in the same dose-dependent manner as the pancreatic one. Immunohistochemical studies revealed that this protease is located in the stromal cells in peri-bronchiolar regions. These results suggest that ectopic anionic trypsin I in rat lungs induced by virus infection may trigger virus spread in rat lungs.     

New insights into the functionality of a virion-anchored host cell membrane protein: CD28 versus HIV type 1

It is now well established that the HIV type 1 (HIV-1) incorporates a vast array of host-encoded molecules in its envelope during the budding process. Interestingly, it was demonstrated that the attachment process is accentuated by supplementary interactions between virion-anchored host molecules and their cognate ligands. Such an enhancement of the viral attachment process was found to result in an increase of infectivity for both T and macrophage-tropic strains of HIV-1. Given that previous work indicates that HIV-1 is budding at the site of cell-to-cell contact, a location rich in the costimulatory CD28 glycoprotein, we investigated whether CD28 could be efficiently acquired by HIV-1. We have been able to generate progeny viruses bearing or not bearing on their surfaces host-derived CD28 using our previously described transient transfection and expression system. The physical presence of CD28 was found to markedly increase virus infectivity in a CD28/B7-dependent manner following infection of two human lymphoid cell lines expressing high levels of surface B7-1/B7-2, two natural ligands of CD28. The physiological significance of CD28 incorporation was provided by the observation that an anti-CD28 Ab decreased replication in primary human mononuclear cells of clinical isolates of HIV-1 propagated in such cells. A virus precipitation assay revealed that M-, T-, and dual-tropic clinical strains of HIV-1 produced in primary human mononuclear cells do indeed incorporate CD28. These results show for the first time that HIV-1 can incorporate CD28 and the acquisition of this specific host surface glycoprotein modulates the virus life cycle.     

Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children’s health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells.Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.     

Influence of Viral Envelope on Newcastle Disease Virus Infection

The adsorption characteristics of Newcastle disease virus (NDV) propagated in chicken cells (NDV-C) and in human cells (NDV-H) were examined. Adsorption experiments performed at different temperatures indicated that virus propagated in a particular cell infected that cell type more readily than did virus propagated in a different host. For example, NDV-C was more efficient in initiating infection of chicken cells at 22 C than was NDV-H; the reverse was true when human cells were employed. The results indicate that infection of susceptible cells by NDV is influenced by the host cell in which the virus was propagated. The data also suggest that NDV may be useful in studies on homologous and heterologous membrane-membrane interactions.     

Experimental Passage of St. Louis Encephalitis Virus In Vivo in Mosquitoes and Chickens Reveals Evolutionarily Significant Virus Characteristics

St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) was the major cause of epidemic flaviviral encephalitis in the U.S. prior to the introduction of West Nile virus (WNV) in 1999. However, outbreaks of SLEV have been significantly more limited then WNV in terms of levels of activity and geographic dispersal. One possible explanation for these variable levels of activity is that differences in the potential for each virus to adapt to its host cycle exist. The need for arboviruses to replicate in disparate hosts is thought to result in constraints on both evolution and host-specific adaptation. If cycling is the cause of genetic stability observed in nature and arboviruses lack host specialization, then sequential passage should result in both the accumulation of mutations and specialized viruses better suited for replication in that host. Previous studies suggest that WNV and SLEV differ in capacity for both genetic change and host specialization, and in the costs each accrues from specializing. In an attempt to clarify how selective pressures contribute to epidemiological patterns of WNV and SLEV, we evaluated mutant spectra size, consensus genetic change, and phenotypic changes for SLEV in vivo following 20 sequential passages via inoculation in either Culex pipiens mosquitoes or chickens. Results demonstrate that the capacity for genetic change is large for SLEV and that the size of the mutant spectrum is host-dependent using our passage methodology. Despite this, a general lack of consensus change resulted from passage in either host, a result that contrasts with the idea that constraints on evolution in nature result from host cycling alone. Results also suggest that a high level of adaptation to both hosts already exists, despite host cycling. A strain significantly more infectious in chickens did emerge from one lineage of chicken passage, yet other lineages and all mosquito passage strains did not display measurable host-specific fitness gains. In addition, increased infectivity in chickens did not decrease infectivity in mosquitoes, which further contrasts the concept of fitness trade-offs for arboviruses.