Pandemic H1N1 Influenza Infection and Vaccination in Humans Induces Cross-Protective Antibodies that Target the Hemagglutinin Stem.

Most monoclonal antibodies (mAbs) generated from humans infected or vaccinated with the 2009 pandemic H1N1 (pdmH1N1) influenza virus targeted the hemagglutinin (HA) stem. These anti-HA stem mAbs mostly used IGHV1-69 and bound readily to epitopes on the conventional seasonal influenza and pdmH1N1 vaccines. The anti-HA stem mAbs neutralized pdmH1N1, seasonal influenza H1N1 and avian H5N1 influenza viruses by inhibiting HA-mediated fusion of membranes and protected against and treated heterologous lethal infections in mice with H5N1 influenza virus. This demonstrated that therapeutic mAbs could be generated a few months after the new virus emerged. Human immunization with the pdmH1N1 vaccine induced circulating antibodies that when passively transferred, protected mice from lethal, heterologous H5N1 influenza infections. We observed that the dominant heterosubtypic antibody response against the HA stem correlated with the relative absence of memory B cells against the HA head of pdmH1N1, thus enabling the rare heterosubtypic memory B cells induced by seasonal influenza and specific for conserved sites on the HA stem to compete for T-cell help. These results support the notion that broadly protective antibodies against influenza would be induced by successive vaccination with conventional influenza vaccines based on subtypes of HA in viruses not circulating in humans.

Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from 1994 to 2008.

H9N2 influenza viruses have been circulating in China since 1994, but a systematic investigation of H9N2 in northern China has not been undertaken since 2004. Here, using the sequences of 22 viruses we isolated from poultry and pigs in northern China during 2003-2008, in combination with sequences available in a public database, we analyzed the evolution of H9N2 influenza viruses in China from 1994 to 2008. Our findings demonstrated that the H9N2 viruses in China underwent extensive reassortment, and novel genotypes continued to emerge. Among 330 viruses, 54 genotypes were observed including 19 novel genotypes that have not been recognized before, and major genotypes were further divided into five series (BJ/94-, G1-, BG-, F/98- and Aq-series). Different epidemiological and biological features among these series were recognized. The BJ/94- and F/98-series viruses were circulating in both southern and northern China, while the other three series viruses were mainly detected in southern China. BJ/94-series influenza viruses predominated in China before 2000 and were gradually replaced by F/98-series viruses that became the predominant viruses since 2004. At least five antigenic groups could be identified over the study period, during which a significant antigenic drift likely occurred between 2002 and 2003. Animal experiments demonstrated that F/98-series viruses were able to replicate and transmit more effectively in chickens than BJ/94-series viruses. The continuing evolution of H9N2 influenza viruses in China emphasizes the importance of H9N2 influenza virus surveillance throughout this region to aid pandemic prediction and prevention.

Prototype single step lateral flow technology for detection of avian influenza virus and chicken antibody to avian influenza virus.

A rapid and effective lateral flow assay (LFA) for detection of avian influenza virus (AIV) was developed. For antigen capture, the assay used monoclonal antibody specific for a conserved nuclear protein (NP) epitope, immobilized on a cellulose acetate matrix, in conjunction with a second NP monoclonal antibody chemically linked to either coloured latex beads or colloidal gold particles contained in a sample pad for detection. Virus sample added to the sample pad flowed into the trapping antibody to form a visible band as well as a second, control band further along the acetate strip. The control band consisted of recombinant protein A/G, also immobilized on the matrix. A second LFA for detection of chicken antibody to AIV was developed where NP antigen was immobilized on the matrix with recombinant protein A/G immobilized as a control band. Latex beads or colloidal gold particles to which monoclonal anti-chicken antibody was attached, were used as the indicator system.

Effect of increasing energy and protein intake on body growth and carcass composition of heifer calves.

The objective was to determine whether increased energy and protein intake between 2 and 14 wk of age would increase growth rates of heifer calves without fattening. At 2 wk of age, Holstein heifer calves were assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement with 2 levels of protein and energy intake (moderate [M]; high [H]) in period 1 (2 to 8 wk of age) by 2 levels of protein and energy intake (low [L]; high [H]) in period 2 (8 to 14 wk of age) to produce similar initial BW for all 4 treatments. Treatments were ML, MH, HL, and HH, indicating moderate or high energy and protein intake during the first period and low or high intake during the second period. The M diet consisted of a standard milk replacer (21.3% CP, 21.3% fat) fed at 1.1% of BW on a DM basis and a 16.5% CP grain mix fed at restricted intake to promote 400 g of average daily gain (ADG), whereas the L diet consisted only of the grain mix. The H diet consisted of a high-protein milk replacer (30.3% CP, 15.9% fat) fed at 2% of BW on a DM basis and a 21.3% CP grain mix available ad libitum. Calves were weaned gradually from milk replacer by 7 wk and slaughtered at 8 (n = 11) or 14 wk of age (n = 41). In periods 1 and 2, ADG and the gain:feed ratio were greater for calves fed the H diet. Calves fed the H diet were taller after both periods 1 and 2. No difference was observed in carcass composition at 8 wk, but at 14 wk calves fed MH and HH had less water and more fat than calves fed ML and HL. Plasma IGF-I concentrations were greatest for calves fed the H diet during either period. Plasma leptin concentrations were increased in calves fed the H diet during period 1 from 4 to 6 wk of age. Increasing energy and protein intake from 2 to 8 wk and 8 to 14 wk of age increased BW, withers height, and gain:feed ratio. Calves fed the H diet from 8 to 14 wk of age had more body fat than calves fed the L diet. Increased energy and protein intake can increase the rate of body growth of heifer calves and potentially reduce rearing costs.

Effect of increasing energy and protein intake on mammary development in heifer calves.

The objective of this study was to determine if increased energy and protein intake from 2 to 14 wk of age would affect mammary development in heifer calves. At 2 wk of age, Holstein heifer calves were assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement with 2 levels of protein and energy intake (moderate, M; high, H) in period 1 (2 to 8 wk of age) and 2 levels of protein and energy intake (low, L; high, H) in period 2 (8 to 14 wk of age), so that mean initial body weights were approximately equal for all 4 treatments (ML, MH, HL, and HH). The M diet in period 1 consisted of a standard milk replacer (21.3% CP, 21.3% fat) fed at 1.1% of BW on a DM basis and a 16.5% CP grain mix fed at restricted intake to promote 400 g of daily gain, whereas the L diet in period 2 consisted only of the grain mix. The H diet in period 1 consisted of a high-protein milk replacer (30.3% CP, 15.9% fat) fed at 2.0% of body weight on a DM basis and a 21.3% CP grain mix available ad libitum. In period 2, the H diet consisted of just the 21.3% grain mix. Calves were gradually weaned from milk replacer by 7 wk and slaughtered at 8 (n = 11) or 14 wk of age (n = 41). Parenchyma from the distal region, midgland, and proximal region relative to the teat from one half of the udder was collected, fixed, and embedded in paraffin. The other half of the gland was used to determine parenchymal mass, protein, fat, DNA, RNA, and extraparenchymal mass. Total parenchymal tissue, parenchymal DNA, parenchymal RNA, and concentrations of DNA and RNA were higher for calves on the H diet during period 1, but were not affected by diet during period 2. Parenchymal fat percentage was increased by the H diet during period 2. The H diet increased extraparenchymal fat during both periods. The area of parenchyma occupied by epithelium was not affected by treatment, but at the end of period 2, the percentage of proliferating epithelial cells as indicated by Ki67, an marker of cell proliferation, expression was greater for calves on the M diet in period 1 compared with calves on the H diet in period 1. Diets did not influence parenchymal protein percentage or the ratio of RNA to DNA. Higher energy and protein intake from 2 to 8 wk of age increased parenchymal mass and parenchymal DNA and RNA in mammary glands of heifer calves without increasing deposition of parenchymal fat. Diet also influenced histological development of mammary parenchyma and subsequent proliferation of ductal epithelial cells. Implications of these effects for future milk production potential are unknown.

Introducing triage logic as a new strategy for the detection of signals in the WHO Drug Monitoring Database.

PURPOSE: An important role for the WHO Programme for International Drug Monitoring is to identify signals of international drug safety problems as early as possible. The signal detection strategy, operated at the Uppsala Monitoring Centre (UMC), gave too many drug-adverse drug reaction (ADR) combinations for individual review. Therefore additional selection strategies were needed to improve the likely signal-to-noise ratio and for the UMC to complement the efforts of national centres in an efficient way. METHODS: The combinations database of the first quarter of 2001 was analysed using algorithms representing different strategies for finding relevant signals using triage logic. RESULTS: The strategies that together gave a manageable number of combinations, i.e. around 600, for further consideration in a single quarter were the algorithms for 'Rapid reporting increase', 'Serious reaction and new drug' and 'Special interests'. These filters began to be used routinely on the combinations database in late 2001. CONCLUSIONS: While stressing that human review is essential, triage strategies are useful when attempting analysis of large amounts of data. By definition, the use of triage strategies may exclude some potential signals from consideration, although the intention is to improve the chances of detection by focussing on areas of greatest importance.

Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: identification of functional themes.

The genetic basis for virulence in influenza virus is largely unknown. To explore the mutational basis for increased virulence in the lung, the H3N2 prototype clinical isolate, A/HK/1/68, was adapted to the mouse. Genomic sequencing provided the first demonstration, to our knowledge, that a group of 11 mutations can convert an avirulent virus to a virulent variant that can kill at a minimal dose. Thirteen of the 14 amino acid substitutions (93%) detected among clonal isolates were likely instrumental in adaptation because of their positive selection, location in functional regions, and/or independent occurrence in other virulent influenza viruses. Mutations in virulent variants repeatedly involved nuclear localization signals and sites of protein and RNA interaction, implicating them as novel modulators of virulence. Mouse-adapted variants with the same hemagglutinin mutations possessed different pH optima of fusion, indicating that fusion activity of hemagglutinin can be modulated by other viral genes. Experimental adaptation resulted in the selection of three mutations that were in common with the virulent human H5N1 isolate A/HK/156/97 and that may be instrumental in its extreme virulence. Analysis of viral adaptation by serial passage appears to provide the identification of biologically relevant mutations.

Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions.

Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigmaNS and core protein mu2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigmaNS and mu2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigmaNS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigmaNS-mutant virus tsE320, sigmaNS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigmaNS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigmaNS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigmaNS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.

The murine double-stranded RNA-dependent protein kinase PKR is required for resistance to vesicular stomatitis virus.

Interferon (IFN)-induced antiviral responses are mediated through a variety of proteins, including the double-stranded RNA-dependent protein kinase PKR. Here we show that fibroblasts derived from PKR(-/-) mice are more permissive for vesicular stomatitis virus (VSV) infection than are wild-type fibroblasts and demonstrate a deficiency in alpha/beta-IFN-mediated protection. We further show that mice lacking PKR are extremely susceptible to intranasal VSV infection, succumbing within days after instillation with as few as 50 infectious viral particles. Again, alpha/beta-IFN was unable to rescue PKR(-/-) mice from VSV infection. Surprisingly, intranasally infected PKR(-/-) mice died not from pathology of the central nervous system but rather from acute infection of the respiratory tract, demonstrating high virus titers in the lungs compared to similarly infected wild-type animals. These results confirm the role of PKR as the major component of IFN-mediated resistance to VSV infection. Since previous reports have shown PKR to be nonessential for survival in animals challenged with encephalomyocarditis virus, influenza virus, and vaccinia virus (N. Abraham et al., J. Biol. Chem. 274:5953-5962, 1999; Y. Yang et al., EMBO J. 14:6095-6106, 1995), our findings serve to highlight the premise that host dependence on the various mediators of IFN-induced antiviral defenses is pathogen specific.

Influenza virus genetics.

Significant progress has been made in understanding the process of influenza A virus replication in cell culture; however, much less is known about the genetic control of virus-host interactions in disease. This review provides an overview of the genetic analysis of influenza virus biology. The functional map of the individual genes of influenza A virus is presented as well as the status of our current understanding of pathogenesis. Influenza has a segmented genome so it is possible to obtain reassortants that contain novel combinations of genome segments derived from different viruses. This is a very useful genetic tool and is also an important aspect of influenza evolution and biology. Human influenza viruses originate from avian strains of influenza virus so that influenza infection is at its basis a zoonosis. Influenza virus strains are host-restricted, however, and avian strains must be adapted to the human host. So questions of host-range and interaction with host factors are important determinants of the ability of influenza virus to cause disease in humans. Host-range is restricted primarily due to host-specific interactions of the ribonucleocapsid and the viral receptor. There are two classes of drugs for inhibiting influenza infection, amantadine HCl and neuraminidase inhibitors. The mode of action and basis for resistance to these drugs are presented. Prospective targets for antiviral therapy are also discussed.

Reovirus mu2 protein determines strain-specific differences in the rate of viral inclusion formation in L929 cells.

Reovirus infection induces the formation of large cytoplasmic inclusions that serve as the major site of viral assembly. Reovirus strains type 3 Dearing (T3D) and type 1 Lang (T1L) differ in the rate of inclusion formation in L929 cells. The median time of inclusion formation is 18 h in cells infected with T3D and 39 h in cells infected with T1L. Using reassortant viruses that contain combinations of gene segments derived from T1L and T3D, we found that the M1 gene, which encodes the mu2 protein, is the primary determinant of the rate of inclusion formation. The S3 gene, which encodes the nonstructural protein sigmaNS, plays a secondary role in this process. The subcellular location of the mu2 protein was determined by confocal laser scanning microscopy using dual-fluorescence labeling of mu2 and the outer-capsid protein mu1/mu1C. In virus-infected cells, mu2 protein colocalized with other viral proteins in inclusions and was also distributed diffusely in the cytoplasm and nucleus. Expression of recombinant T1L and T3D mu2 proteins resulted in the formation of protein complexes resembling inclusions in both the cytoplasm and the nucleus with kinetics that reflected the strain of origin. The median time of mu2 protein complex formation was 22 h in cells transfected with the T3D M1 gene and 43 h in cells transfected with the T1L M1 gene. These findings suggest that the mu2 protein influences the rate of inclusion formation and contributes to inclusion morphogenesis. The requirement of mu2 protein in inclusion formation was tested by determining the subcellular localization of mu2 in cells infected with temperature-sensitive (ts) mutants that are defective in viral assembly. In contrast to infection with wild-type virus, mu2 did not colocalize with mu1/mu1C protein in subcellular structures that formed in cells infected at nonpermissive temperature with ts mutants tsH11.2, tsC447, and tsG453 with mutations in the M1, S2, and S4 genes, respectively. These results suggest that despite the role of the mu2 protein in controlling the rate of inclusion formation, this process is a concerted function of several reovirus proteins.

Evaluation of a neonatal rat model for prediction of mumps virus neurovirulence in humans.

Neurovirulence of several mumps virus strains was assessed in a prototype rat neurovirulence test and compared to results obtained in the monkey neurovirulence test. The relative human neurovirulence of these strains was proportional to the severity of hydrocephalus in rats but not to lesion scores in the monkeys.

Heteroduplex mobility assay-guided sequence discovery: elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools.

The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA "signature" sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus.

The Urabe AM9 mumps vaccine is composed of a mixture of variants distinguishable by a difference at nucleotide (nt) 1081 of the hemagglutinin-neuraminidase (HN) gene (Brown, E.G., Dimock, K., Wright, K.E., 1996. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid (aa) 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J. Infect. Dis. 174, 619-622.). Further genetic and biological variation was detected in plaque purified viruses from the Urabe AM9 vaccine by examining the HN gene sequence, plaque morphology, cytopathic effects and growth in Vero cells, and temperature sensitivity (ts). Infection of Vero cells with plaque purified viruses with a G at nt 1081 of the HN gene produced large, clear plaques, caused significant CPE early after infection but yielded lower titres of virus than other purified viruses. None of these viruses were ts. In contrast, half of the plaque purified viruses with an A at nt 1081 were sensitive to a temperature of 39.5 degrees C. These viruses produced small plaques, caused significant CPE and grew to low titres. Two ts viruses possessed a unique aa substitution at aa 468 of HN. The remaining A(1081) viruses were not ts, produced large plaques but little CPE, and grew to titres 10-fold higher than the G(1081) viruses. Isolates of Urabe AM9 associated with post-vaccination illness were similar to these non-ts A(1081) viruses, but could be further sub-divided into two groups on the basis of a difference at aa 464 of HN. The post-vaccination isolates may represent insufficiently attenuated components of the vaccine, while the G(1081) and ts subset of A(1081) viruses may be more fully attenuated.

Genetic analysis of mouse-adapted influenza A virus identifies roles for the NA, PB1, and PB2 genes in virulence.

Adaptation of the prototype A/FM/1/47 H1N1 strain to mice resulted in selection of the A/FM/1/47-MA variant with increased virulence. Earlier analysis identified mutations in the HA and M1 genes that increase virulence in the mouse. Complete sequence analysis identified mutations in the PB1, PB2, HA, NA, and M1 genes. Reassortants were produced between the parental FM and FM-MA strains to obtain viruses that differ due to combinations of mutant genes. To assess the relationship between virulence and replication, the median lethal dose was determined for mice and growth properties were assessed in mouse lung, MDCK cells and chicken embryo. Not only were all five mutations shown to control virulence but also the replicative capacity in the mouse. The HA, NA and M1 mutations increased yield in all three hosts whereas in combination the PB1 and PB2 mutations were host restrictive changing the virus to a mouse specific strain. For the NA and M1 mutations the increase in growth in mouse lung was proportional to a 2-fold (log10) increase in virulence however the HA mutation increased virulence largely independent of increased growth indicating a change in pathological properties that damage the host. Thus mutations that affect virulence can be classified according to host-dependent and independent ability to increase growth as well as changes in pathological properties. Each of the PB1, PB2, NA, HA, and M1 genes acquired gain-of-function mutations for mouse infection that involve structural motifs that may serve as markers for virulence or targets for antiviral therapy.

The mumps virus neurovirulence safety test in Rhesus monkeys: a comparison of mumps virus strains.

Wild type mumps viruses are highly neurotropic and a frequent cause of aseptic meningitis in unvaccinated humans. To test whether attenuated mumps viruses used in the manufacture of mumps vaccines have neurovirulent properties, a monkey neurovirulence safety test (MNVT) is performed. However, results with several mumps virus MNVTs have raised questions as to whether the test can reliably discriminate neurovirulent from nonneurovirulent mumps virus strains. Here, various mumps virus strains representing a wide range of neuropathogenicity were tested in a standardized MNVT. A trend of higher neurovirulence scores was observed in monkeys inoculated with wild type mumps virus versus vaccine strains, although differences were not statistically significant. Results indicated the need for further examination and refinement of the MNVT or for development of alternative MNVTs.

Immunochemical localization of platelet-derived growth factor in placenta and its possible role in pre-eclampsia.

BACKGROUND: Platelet-derived growth factor (PDGF) has an important role in cellular function, proliferation, and angiogenesis. It is also associated with tissue injury and repair, and its involvement in atherosclerosis has been studied extensively. Tissue injury is also found in pre-eclampsia (PRE). The morphological and physiological changes that are taking place in the PRE placenta may be associated with this growth factor. The objective of this study was to determine if PDGF-AA and its alpha receptor are present in the cells and vessels of normotensive (NORM) and PRE placentas at term. METHODS: Placental tissue was obtained from 6 preeclamptic and 8 normotensive women. Gestational age ranged from 38-42 weeks. Deliveries were either vaginal (vag, n = 10) or c-section (c-sec, n = 4). Tissue samples were analyzed for PDGF-AA by immunocytochemistry. RESULTS: PDGF-AA and the alpha receptor were present in both NORM and PRE placentas. Immunoreactive staining revealed PDGF-AA and its receptor in the intimal/endothelial layer of fetal vessels and the trophoblastic layer. Staining intensity was greater in preeclamptic tissue when obliterative endarteritis was present. CONCLUSIONS: In pre-eclampsia, PDGF-AA may play a role in the restructuring of the fetoplacental vasculature, in particular when there is inflammation of the vascular intimal layer, as found in obliterative endarteritis. Increased staining in the trophoblast layer in patients with obliterative endarteritis also may be indicative of more widespread damage throughout the placenta itself, and PDGF-AA may play a significant role in the repair of this damage.

The medical dictionary for regulatory activities (MedDRA).

The International Conference on Harmonisation has agreed upon the structure and content of the Medical Dictionary for Regulatory Activities (MedDRA) version 2.0 which should become available in the early part of 1999. This medical terminology is intended for use in the pre- and postmarketing phases of the medicines regulatory process, covering diagnoses, symptoms and signs, adverse drug reactions and therapeutic indications, the names and qualitative results of investigations, surgical and medical procedures, and medical/social history. It can be used for recording adverse events and medical history in clinical trials, in the analysis and tabulations of data from these trials and in the expedited submission of safety data to government regulatory authorities, as well as in constructing standard product information and documentation for applications for marketing authorisation. After licensing of a medicine, it may be used in pharmacovigilance and is expected to be the preferred terminology for international electronic regulatory communication. MedDRA is a hierarchical terminology with 5 levels and is multiaxial: terms may exist in more than 1 vertical axis, providing specificity of terms for data entry and flexibility in data retrieval. Terms in MedDRA were derived from several sources including the WHO's adverse reaction terminology (WHO-ART), Coding Symbols for a Thesaurus of Adverse Reaction Terms (COSTART), International Classification of Diseases (ICD) 9 and ICD9-CM. It will be maintained, further developed and distributed by a Maintenance Support Services Organisation (MSSO). It is anticipated that using MedDRA will improve the quality of data captured on databases, support effective analysis by providing clinically relevant groupings of terms and facilitate electronic communication of data, although as a new tool, users will need to invest time in gaining expertise in its use.

Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR.

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s).