SARS-CoV-2 has undergone repeated and rapid evolution to circumvent host immunity. However, outside of prolonged infections in immunocompromised hosts, within-host positive selection has rarely been detected. Here we combine daily longitudinal sampling of individuals with replicate sequencing to increase the accuracy of and lower the threshold for variant calling. We sequenced 577 specimens from 105 individuals in a household cohort during the BA.1/BA.2 variant period. Individuals exhibited extremely low viral diversity, and we estimated a low within-host evolutionary rate. Within-host dynamics were dominated by genetic drift and purifying selection. Positive selection was rare but highly concentrated in spike. A Wright Fisher Approximate Bayesian Computational model identified positive selection at 14 loci with 7 in spike, including S:448 and S:339. This detectable immune-mediated selection is unusual in acute respiratory infections and may be caused by the relatively narrow antibody repertoire in individuals during the early Omicron phase of the SARS-CoV-2 pandemic.

BACKGROUND:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic and subgenomic RNA levels are frequently used as a correlate of infectiousness. The impact of host factors and SARS-CoV-2 lineage on RNA viral load is unclear.METHODS:Total nucleocapsid (N) and subgenomic N (sgN) RNA levels were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in specimens from 3204 individuals hospitalized with coronavirus disease 2019 (COVID-19) at 21 hospitals. RT-qPCR cycle threshold (Ct) values were used to estimate RNA viral load. The impact of time of sampling, SARS-CoV-2 variant, age, comorbidities, vaccination, and immune status on N and sgN Ct values were evaluated using multiple linear regression.RESULTS:Mean Ct values at presentation for N were 24.14 (SD 4.53) for non-variants of concern, 25.15 (SD 4.33) for Alpha, 25.31 (SD 4.50) for Delta, and 26.26 (SD 4.42) for Omicron. N and sgN RNA levels varied with time since symptom onset and infecting variant but not with age, comorbidity, immune status, or vaccination. When normalized to total N RNA, sgN levels were similar across all variants.CONCLUSIONS:RNA viral loads were similar among hospitalized adults, irrespective of infecting variant and known risk factors for severe COVID-19. Total N and subgenomic RNA N viral loads were highly correlated, suggesting that subgenomic RNA measurements add little information for the purposes of estimating infectivity.

OBjECTIVES To determine the association between covid-19 vaccination types and doses with adverse outcomes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection during the periods of delta (B.1.617.2) and omicron (B.1.1.529) variant predominance.DESIGN Retrospective cohort.SETTING US Veterans Affairs healthcare system.PARTICIPANTS Adults (>= 18 years) who are affiliated to Veterans Affairs with a first documented SARS-CoV-2 infection during the periods of delta (1 July-30 November 2021) or omicron (1 January-30 June 2022) variant predominance. The combined cohorts had a mean age of 59.4 (standard deviation 16.3) and 87% were male. INTERVENTIONS Covid-19 vaccination with mRNA vaccines (BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna)) and adenovirus vector vaccine (Ad26.COV2.S (Janssen/ Johnson & Johnson)).MAIN OUTCOME MEASURES Stay in hospital, intensive care unit admission, use of ventilation, and mortality measured 30 days after a positive test result for SARS-CoV-2.RESULTS In the delta period, 95 336 patients had infections with 47.6% having at least one vaccine dose, compared with 184 653 patients in the omicron period, with 72.6% vaccinated. After adjustment for patient demographic and clinical characteristics, in the delta period, two doses of the mRNA vaccines were associated with lower odds of hospital admission (adjusted odds ratio 0.41 (95% confidence interval 0.39 to 0.43)), intensive care unit admission (0.33 (0.31 to 0.36)), ventilation (0.27 (0.24 to 0.30)), and death (0.21 (0.19 to 0.23)), compared with no vaccination. In the omicron period, receipt of two mRNA doses were associated with lower odds of hospital admission (0.60 (0.57 to 0.63)), intensive care unit admission (0.57 (0.53 to 0.62)), ventilation (0.59 (0.51 to 0.67)), and death (0.43 (0.39 to 0.48)). Additionally, a third mRNA dose was associated with lower odds of all outcomes compared with two doses: hospital admission (0.65 (0.63 to 0.69)), intensive care unit admission (0.65 (0.59 to 0.70)), ventilation (0.70 (0.61 to 0.80)), and death (0.51 (0.46 to 0.57)). The Ad26.COV2.S vaccination was associated with better outcomes relative to no vaccination, but higher odds of hospital stay and intensive care unit admission than with two mRNA doses. BNT162b2 was generally associated with worse outcomes than mRNA-1273 (adjusted odds ratios between 0.97 and 1.42).CONCLUSIONS In veterans with recent healthcare use and high occurrence of multimorbidity, vaccination was robustly associated with lower odds of 30 day morbidity and mortality compared with no vaccination among patients infected with covid-19. The vaccination type and number of doses

Monoclonal antibody treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been widely implemented. Effects of treatment on the endogenous primary humoral response to the virus are unknown. A retrospective cohort study performed at a Veterans Health Administration medical center compared serologic responses of treated and untreated COVID-19 patients at high risk for severe outcomes. Three anti-viral spike protein IgG monoclonal treatments were used during the study period, 1) bamlanivimab, 2) casirivimab with imdevimab, and 3) bamlanivimab with etesevimab. Data were analyzed at acute (0-9 days), seroconversion (10-19 days), and maximum antibody (20-39 days) stages. SARS-Cov-2 infection induced a dynamic primary humoral response with anti-spike IgM and anti-nucleocapsid IgG seroconversion occurring after 9 days with maximum serologic indices achieved by 20-39 days. All monoclonal antibody treatments suppressed the endogenous anti-spike IgM response by 85-90% with minor effect on the anti-nucleocapsid response. Thus, passive immunization therapy may cause immunologic interference.

SARS-CoV-2 mRNA vaccines have been critical to curbing pandemic COVID-19; however, a major shortcoming has been the inability to assess levels of protection after vaccination. This study assessed serologic status of breakthrough infections in vaccinated patients at a Veterans Administration medical center from June through December 2021 during a SARS-CoV-2 delta variant wave. Breakthrough occurred mostly beyond 150 days after two-dose vaccination with a mean of 239 days. Anti-SARS-CoV-2 spike (S) IgG levels were low at 0 to 2 days postsymptoms but increased in subjects presenting thereafter. Population measurements of anti-S IgG and angiotensin converting enzyme-2 receptor (ACE2-R) binding inhibition among uninfected, vaccinated patients suggested immune decay occurred after 150 days with 62% having anti-S IgG levels at or below 1,000 AU comparable with breakthrough patients at 0 to 2 days postsymptom onset. In contrast, vaccination after resolved infection conferred robust enduring anti-S IgG levels (5,000 to >50,000 AU) with >90% ACE2-R binding inhibition. However, monoclonal antibody (MAb)-treated patients did not benefit from their prior infection suggesting impaired establishment of B cell memory. Analysis of boosted patients confirmed the benefit of a third vaccine dose with most having anti-S IgG levels above 5,000 AU with >90% ACE2-R binding inhibition, but a subset had levels <5,000 AU. Anti-S IgG levels >5,000 AU were associated with >90% ACE2-R binding inhibition and no documented breakthrough infections, whereas levels falling below 5,000 AU and approaching 1,000 AU were associated with breakthrough infections. Thus, quantitative antibody measurements may provide a means to guide vaccination intervals for the individual. IMPORTANCE Currently, clinicians have no guidance for the serologic assessment of SARS-Cov-2 postvaccination status regarding protection and risk of infection. Vaccination and boosters are administered blindly without evaluation of need or outcome at the individual level. The recent development of automated quantitative assays for anti-SARS-CoV-2 spike protein IgG antibodies permits accurate measurement of humoral immunity in standardized units. Clinical studies, such as reported here, will help establish protective antibody levels allowing identification and targeted management of poor vaccine responders and vaccinated subjects undergoing immune decay.

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified viral load as a critical factor in variant identification. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.

Understanding viral load in patients infected with SARS-CoV-2 is critical to epidemiology and infection control. Previous studies have demonstrated that SARS-CoV-2 RNA can be detected for many weeks after symptom onset. The clinical significance of this finding is unclear and, in most patients, likely does not represent active infection. There are, however, patients who shed infectious virus for weeks. Detection of subgenomic RNA transcripts expressed by SARS-CoV-2 has been proposed to represent productive infection and may be a tractable marker for monitoring infectivity. Here, we use RT-PCR to quantify total and subgenomic nucleocapsid (N) and envelope (E) transcripts in 190 SARS-CoV-2 positive samples collected on hospital admission. We relate these findings to duration of symptoms. We find that all transcripts decline at the same rate; however, subgenomic E becomes undetectable before other transcripts. In Kaplan-Meier analysis the median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic RNA compared to total RNA suggesting subgenomic transcript copy number is highly dependent on copy number of total transcripts. The mean difference between total N and subgenomic N is 16-fold (4.0 cycles) and the mean difference between total E and sub-genomic E is 137-fold (7.1 cycles). This relationship is constant over duration of symptoms allowing prediction of subgenomic copy number from total copy number. Although Subgenomic E is undetectable at a time that may more closely reflect the duration of infectivity, its utility in determining active infection may be no more useful than a copy number threshold determined for total transcripts.

BACKGROUND:Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS:We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS:We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS:Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Objective: The seroprevalence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) IgG antibody was evaluated among employees of a Veterans Affairs healthcare system to assess potential risk factors for transmission and infection. Methods: All employees were invited to participate in a questionnaire and serological survey to detect antibodies to SARS-CoV-2 as part of a facility-wide quality improvement and infection prevention initiative regardless of clinical or nonclinical duties. The initiative was conducted from June 8 to July 8, 2020. Results: Of the 2,900 employees, 51% participated in the study, revealing a positive SARS-CoV-2 seroprevalence of 4.9% (72 of 1,476; 95% CI, 3.8%-6.1%). There were no statistically significant differences in the presence of antibody based on gender, age, frontline worker status, job title, performance of aerosol-generating procedures, or exposure to known patients with coronavirus infectious disease 2019 (COVID-19) within the hospital. Employees who reported exposure to a known COVID-19 case outside work had a significantly higher seroprevalence at 14.8% (23 of 155) compared to those who did not 3.7% (48 of 1,296; OR, 4.53; 95% CI, 2.67-7.68; P < .0001). Notably, 29% of seropositive employees reported no history of symptoms for SARS-CoV-2 infection. Conclusions: The seroprevalence of SARS-CoV-2 among employees was not significantly different among those who provided direct patient care and those who did not, suggesting that facility-wide infection control measures were effective. Employees who reported direct personal contact with COVID-19-positive persons outside work were more likely to have SARS-CoV-2 antibodies. Employee exposure to SARS-CoV-2 outside work may introduce infection into hospitals.

IMPORTANCE:The association between red blood cell (RBC) transfusion strategies and health care-associated infection is not fully understood. OBJECTIVE:To evaluate whether RBC transfusion thresholds are associated with the risk of infection and whether risk is independent of leukocyte reduction. DATA SOURCES:MEDLINE, EMBASE, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, Cochrane Database of Sytematic Reviews, ClinicalTrials.gov, International Clinical Trials Registry, and the International Standard Randomized Controlled Trial Number register were searched through January 22, 2014. STUDY SELECTION:Randomized clinical trials with restrictive vs liberal RBC transfusion strategies. DATA EXTRACTION AND SYNTHESIS:Twenty-one randomized trials with 8735 patients met eligibility criteria, of which 18 trials (n = 7593 patients) contained sufficient information for meta-analyses. DerSimonian and Laird random-effects models were used to report pooled risk ratios. Absolute risks of infection were calculated using the profile likelihood random-effects method. MAIN OUTCOMES AND MEASURES:Incidence of health care-associated infection such as pneumonia, mediastinitis, wound infection, and sepsis. RESULTS:The pooled risk of all serious infections was 11.8% (95% CI, 7.0%-16.7%) in the restrictive group and 16.9% (95% CI, 8.9%-25.4%) in the liberal group. The risk ratio (RR) for the association between transfusion strategies and serious infection was 0.82 (95% CI, 0.72-0.95) with little heterogeneity (I2 = 0%; τ2 <.0001). The number needed to treat (NNT) with restrictive strategies to prevent serious infection was 38 (95% CI, 24-122). The risk of infection remained reduced with a restrictive strategy, even with leukocyte reduction (RR, 0.80 [95% CI, 0.67-0.95]). For trials with a restrictive hemoglobin threshold of <7.0 g/dL, the RR was 0.82 (95% CI, 0.70-0.97) with NNT of 20 (95% CI, 12-133). With stratification by patient type, the RR was 0.70 (95% CI, 0.54-0.91) in patients undergoing orthopedic surgery and 0.51 (95% CI, 0.28-0.95) in patients presenting with sepsis. There were no significant differences in the incidence of infection by RBC threshold for patients with cardiac disease, the critically ill, those with acute upper gastrointestinal bleeding, or for infants with low birth weight. CONCLUSIONS AND RELEVANCE:Among hospitalized patients, a restrictive RBC transfusion strategy was associated with a reduced risk of health care-associated infection compared with a liberal transfusion strategy. Implementing restrictive strategies may have the potential to lower the incidence of health care-associated infection.

Importance The association between red blood cell (RBC) transfusion strategies and health care–associated infection is not fully understood. Objective To evaluate whether RBC transfusion thresholds are associated with the risk of infection and whether risk is independent of leukocyte reduction. Data Sources MEDLINE, EMBASE, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, Cochrane Database of Sytematic Reviews, ClinicalTrials.gov, International Clinical Trials Registry, and the International Standard Randomized Controlled Trial Number register were searched through January 22, 2014. Study Selection Randomized clinical trials with restrictive vs liberal RBC transfusion strategies. Data Extraction and Synthesis Twenty randomized trials with 8598 patients met eligibility criteria, of which 17 trials (n = 7456 patients) contained sufficient information for meta-analyses. DerSimonian and Laird random-effects models were used to report pooled risk ratios. Absolute risks of infection were calculated using the profile likelihood random-effects method. Main Outcomes and Measures Incidence of health care–associated infection such as pneumonia, mediastinitis, wound infection, and sepsis. Results The pooled risk of all serious infections was 10.6% (95% CI, 5.6%-15.9%) in the restrictive group and 12.7% (95% CI, 7.0%-18.7%) in the liberal group. The risk ratio (RR) for the association between transfusion strategies and infection (serious infections and selected infections, combined) was 0.92 (95% CI, 0.82-1.04) with little heterogeneity (I2 = 6.3%; τ2 = .0041). The RR for the association between transfusion strategies and serious infection was 0.84 (95% CI, 0.73-0.96;I2 = 0%, τ2<.0001). The number needed to treat (NNT) with restrictive strategies to prevent serious infection was 48 (95% CI, 36-71). The risk of infection remained reduced with a restrictive strategy, even with leukocyte reduction (RR, 0.83 [95% CI, 0.69-0.99]). For trials with a restrictive hemoglobin threshold of <7.0 g/dL, the RR was 0.86 (95% CI, 0.72-1.02). With stratification by patient type, the RR for serious infection was 0.72 (95% CI, 0.53-0.97) in patients undergoing orthopedic surgery and 0.51 (95% CI, 0.28-0.95) in patients presenting with sepsis. There were no significant differences in the incidence of infection by RBC threshold for patients with cardiac disease, the critically ill, those with acute upper gastrointestinal bleeding, or for infants with low birth weight. Conclusions and Relevance Among hospitalized patients, a restrictive RBC transfusion strategy compared with a liberal transfusion strategy was not associated with a reduced risk of health care–associated infection overall, although it was associated with a reduced risk of serious infection. Implementing restrictive strategies may have the potential to lower the incidence of serious health care–associated infection.

Practitioners of hospital medicine should be familiar with the indications and complications of red blood cell (RBC) transfusions. Recent evidence based guidelines support the widespread use of a restrictive RBC transfusion strategy. Not only does a restrictive transfusion strategy reduce the amount of RBCs used, but it also has been associated with improved patient outcomes. Clinicians should understand how to recognize and effectively manage complications of RBC transfusions. With improved screening techniques, direct infections from RBC transfusions have become less common than noninfectious complications such as transfusion-related acute lung injury, febrile non-hemolytic transfusion reaction, and acute hemolytic reactions.

The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic β subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the β subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative β sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.

BACKGROUND:Certain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform motor neuron disease in susceptible mice upon infection of the central nervous system (CNS). The major CNS parenchymal target of these neurovirulent retroviruses (NVs) are the microglia, whose infection is largely coincident with neuropathological changes. Despite this close association, the role of microglial infection in disease induction is still unknown. In this paper, we investigate the interaction of the highly virulent MLV, FrCasE, with microglia ex vivo to evaluate whether infection induces specific changes that could account for neurodegeneration. Specifically, we compared microglia infected with FrCasE, a related non-neurovirulent virus (NN) F43/Fr57E, or mock-infected, both at a basic virological level, and at the level of cellular gene expression using quantitative real time RT-PCR (qRT-PCR) and Afffymetrix 430A mouse gene chips. RESULTS:Basic virological comparison of NN, NV, and mock-infected microglia in culture did not reveal differences in virus expression that provided insight into neuropathogenesis. Therefore, microglial analysis was extended to ER stress gene induction based on previous experiments demonstrating ER stress induction in NV-infected mouse brains and cultured fibroblasts. Analysis of message levels for the ER stress genes BiP (grp78), CHOP (Gadd153), calreticulin, and grp58 in cultured microglia, and BiP and CHOP in microglia enriched fractions from infected mouse brains, indicated that FrCasE infection did not induce these ER stress genes either in vitro or in vivo. To broadly identify physiological changes resulting from NV infection of microglia in vitro, we undertook a gene array screen of more than 14,000 well-characterized murine genes and expressed sequence tags (ESTs). This analysis revealed only a small set of gene expression changes between infected and uninfected cells (<18). Remarkably, gene array comparison of NN- and NV-infected microglia revealed only 3 apparent gene expression differences. Validation experiments for these genes by Taqman real-time RT-PCR indicated that only single Ig IL-1 receptor related protein (SIGIRR) transcript was consistently altered in culture; however, SIGIRR changes were not observed in enriched microglial fractions from infected brains. CONCLUSION:The results from this study indicate that infection of microglia by the highly neurovirulent virus, FrCasE, does not induce overt physiological changes in this cell type when assessed ex vivo. In particular, NV does not induce microglial ER stress and thus, FrCasE-associated CNS ER stress likely results from NV interactions with another cell type or from neurodegeneration directly. The lack of NV-induced microglial gene expression changes suggests that FrCasE either affects properties unique to microglia in situ, alters the expression of microglial genes not represented in this survey, or affects microglial cellular processes at a post-transcriptional level. Alternatively, NV-infected microglia may simply serve as an unaffected conduit for persistent dissemination of virus to other neural cells where they produce acute neuropathogenic effects.

The neurovirulent retroviruses FrCasE and Moloney MLV-ts1 cause noninflammatory spongiform neurodegeneration in mice, manifested clinically by progressive spasticity and paralysis. Neurons have been thought to be the primary target of toxicity of these viruses. However the neurons themselves appear not to be infected, and the possible indirect mechanisms driving the neuronal toxicity have remained enigmatic. Here we have re-examined the cells that are damaged by these viruses, using lineage-specific markers. Surprisingly, these cells expressed the basic helix-loop-helix transcription factor Olig2, placing them in the oligodendrocyte lineage. Olig2+ cells were found to be infected, and many of these cells exhibited focal cytoplasmic vacuolation, suggesting that infection by spongiogenic retroviruses is directly toxic to these cells. As cytoplasmic vacuolation progressed, however, signs of viral protein expression appeared to wane, although residual viral RNA was detectable by in situ hybridization. Cells with the most advanced cytoplasmic effacement expressed the C/EBP-homologous protein (CHOP). This protein is up-regulated as a late event in a cellular response termed the integrated stress response. This observation may link the cellular pathology observed in the brain with cellular stress responses known to be induced by these viruses. The relevance of these observations to oligodendropathy in humans is discussed.

Data regarding the occurrence of a viremia during rabies virus infections are contradictory. Here, we attempted to clarify the dissimilar results using a qualitative TaqMan PCR assay to detect viral RNA in blood of mice that had been injected intramuscularly with rabies virus. Viral RNA was detected at two different intervals. Initially, RNA was present in blood of 30/32 (94%) mice, from 1h to 2 days after injection of virus. The RNA in the blood at this time most likely resulted from trauma to blood vessels at the injection site and leakage of the inoculated virus into the circulation. Thereafter, from 3 to 30 days, viral RNA was undetectable in the blood of 37 mice that remained free of clinical disease. However, and more importantly, viral RNA was detected again in 21/25 (84%) mice that became clinically ill and were exsanguinated 2–4 days after the onset of paralysis. The presence of viral RNA in blood of the clinically ill mice might have been due to an escape of virus into the bloodstream as a result of viral replication induced injury in the central nervous system and other tissues. Anti-rabies virus neutralizing antibody was detected in sera of 11/21 (52%) clinically ill mice whose blood was positive for rabies viral RNA. The presence of viral RNA in the bloodstream of mice that developed clinical rabies suggested that a viremia might occur in rabies-infected mice. Thus, the current opinion that a viremia does not occur in experimental or natural rabies infections of other species might need to be re-evaluated.

Several different groups of birds have been proposed as being the oldest or earliest diverging extant lineage within the avian phylogenetic tree, particularly ratites (Struthioniformes), waterfowl (Anseriformes), and shorebirds (Charadriiformes). Difficulty in resolving this issue stems from several factors, including the relatively rapid radiation of primary (ordinal) bird lineages and the lack of characters from an extant outgroup for birds that is closely related to them by measure of time. To help resolve this question, we have sequenced entire mitochondrial genomes for five birds (a rhea, a duck, a falcon, and two perching birds), one crocodilian, and one turtle. Maximum parsimony and maximum likelihood analyses of these new sequences together with published sequences (18 taxa total) yield the same optimal tree topology, in which a perching bird (Passeriformes) is sister to all the other bird taxa. A basal position for waterfowl among the bird study taxa is rejected by maximum likelihood analyses. However, neither the conventional view, in which ratites (including rhea) are basal to other birds, nor tree topologies with falcon or chicken basal among birds could be rejected in the same manner. In likelihood analyses of a subset of seven birds, alligator, and turtle (9 taxa total), we find that increasing the number of parameters in the model shifts the optimal topology from one with a perching bird basal among birds to the conventional view with ratites diverging basally; moreover, likelihood scores for the two trees are not significantly different. Thus, although our largest set of taxa and characters supports a tree with perching birds diverging basally among birds, the position of this earliest divergence among birds appears unstable. Our analyses indicate a sister relationship between a waterfowl/chicken clade and ratites, relative to perching birds and falcon. We find support for a sister relationship between turtles and a bird/crocodilian clade, and for rejecting both the Haemothermia hypothesis (birds and mammals as sister taxa) and the placement of turtles as basal within the phylogenetic tree for amniote animals.