Cellulitis and bacteremia caused by the fish pathogen,Streptococcus iniae, in an immunocompromised patient: Case report and mini-review of zoonotic disease, lab identification, and antimicrobial susceptibility.

Streptococcus iniae is a fish pathogen that can also infect mammals including dolphins and humans. Its prevalence in farmed fish, particularly tilapia, provides potential for zoonotic infections, as documented by multiple case reports. Systematic clinical data beyond cellulitis for S. iniae infection in humans, including antimicrobial susceptibility data, are unfortunately rare. Here, we present a case of cellulitis progressing to bacteremia caused by Streptococcus iniae in a functionally immunocompromised patient based on CDK4/CDK6 inhibitor and endocrine therapy, and we discuss risk factors, identification, and antimicrobial susceptibility of this rare pathogen.

Case report and mini-review: Sarcina ventriculi in the stomach of an 80-year-old female.

Sarcina ventriculi, also known as Zymosarcina ventriculi and, incorrectly, as Clostridium ventriculi, is rarely encountered in clinical settings. A patient with a complicated gastrointestinal (GI) history, who was acutely presenting with small-bowel obstruction, was found to be colonized by S. ventriculi. The distinctive morphology of this species, with large Gram-variable cocci (up to 3 µm) arranged in two-by-two cuboid clusters reaching up to 20 µm, was key in identifying this bacterium in a stomach biopsy specimen. Sarcina ventriculi appears to be ubiquitously found in nature, and related bacterial species can cause GI-related disease in various animals. Clinical manifestations in humans are broad and often related to other underlying comorbidities. Isolation of S. ventriculi in the laboratory requires anaerobic culture on select media but its absence from standard MALDI-TOF databases complicates identification. Susceptibility data do not exist, so empiric treatment is the only option for this rare pathogen.

Universal PCR for bacteria, mycobacteria, and fungi: a 10-year retrospective review of clinical indications and patient outcomes.

IMPORTANCE: Our work provides a retrospective analysis of universal PCR orders for bacteria, mycobacteria, and fungi across our institution across a 10-year period. We assessed the positivity rates for this diagnostic tool by test type and specimen type and, critically, studied whether and how the results influenced the outcomes from treatment change, to readmission, to death.

APOBEC3F Constitutes a Barrier to Successful Cross-Species Transmission of Simian Immunodeficiency Virus SIVsmm to Humans.

Simian immunodeficiency virus infecting sooty mangabeys (SIVsmm) has been transmitted to humans on at least nine occasions, giving rise to human immunodeficiency virus type 2 (HIV-2) groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in sub-Saharan Africa, and it is largely unclear whether adaptive changes were associated with spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains and representatives of five different SIVsmm lineages to various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in human T cells, albeit with more variable replication fitness and frequently lower efficiency than HIV-2 IMCs. Efficient viral propagation was generally dependent on intact vif genes, highlighting the need for counteraction of APOBEC3 proteins. On average, SIVsmm was more susceptible to inhibition by human APOBEC3D, -F, -G, and -H than HIV-2. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by ∼80% but achieved only ∼40% reduction in the case of HIV-2. Functional and mutational analyses of human- and monkey-derived alleles revealed that an R128T polymorphism in APOBEC3F contributes to species-specific counteraction by HIV-2 and SIVsmm Vifs. In addition, a T84S substitution in SIVsmm Vif increased its ability to counteract human APOBEC3F. Altogether, our results confirm that SIVsmm Vif proteins show intrinsic activity against human APOBEC3 proteins but also demonstrate that epidemic HIV-2 strains evolved an increased ability to counteract this class of restriction factors during human adaptation. IMPORTANCE Viral zoonoses pose a significant threat to human health, and it is important to understand determining factors. SIVs infecting great apes gave rise to HIV-1. In contrast, SIVs infecting African monkey species have not been detected in humans, with one notable exception. SIVsmm from sooty mangabeys has crossed the species barrier to humans on at least nine independent occasions and seems capable of overcoming many innate defense mechanisms without adaptation. Here, we confirmed that SIVsmm Vif proteins show significant activity against human APOBEC3 proteins. Our analyses also revealed, however, that different lineages of SIVsmm are significantly more susceptible to inhibition by various human APOBEC3 proteins than HIV-2 strains. Mutational analyses suggest that an R128T substitution in APOBEC3F and a T84S change in Vif contribute to species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that epidemic HIV-2 strains acquired increased activity against human APOBEC3 proteins to clear this restrictive barrier.

SIV infection duration largely determines broadening of neutralizing antibody response in macaques.

The development of broadly neutralizing antibodies (BNAbs) in HIV infection is a result of long-term coevolutionary interaction between viruses and antibodies. Understanding how this interaction promotes the increase of neutralization breadth during infection will improve the way in which AIDS vaccine strategies are designed. In this paper, we used SIV-infected rhesus macaques as a model to study the development of neutralization breadth by infecting rhesus macaques with longitudinal NAb escape variants and evaluating the kinetics of NAb response and viral evolution. We found that the infected macaques developed a stepwise NAb response against escape variants and increased neutralization breadth during the course of infection. Furthermore, the increase of neutralization breadth correlated with the duration of infection but was independent of properties of the inoculum, viral loads, or viral diversity during infection. These results imply that the duration of infection was the main factor driving the development of BNAbs. These data suggest the importance of novel immunization strategies to induce effective NAb response against HIV infection by mimicking long-term infection.

TRIM5α Resistance Escape Mutations in the Capsid Are Transferable between Simian Immunodeficiency Virus Strains.

TRIM5α polymorphism limits and complicates the use of simian immunodeficiency virus (SIV) for evaluation of human immunodeficiency virus (HIV) vaccine strategies in rhesus macaques. We previously reported that the TRIM5α-sensitive SIV from sooty mangabeys (SIVsm) clone SIVsmE543-3 acquired amino acid substitutions in the capsid that overcame TRIM5α restriction when it was passaged in rhesus macaques expressing restrictive TRIM5α alleles. Here we generated TRIM5α-resistant clones of the related SIVsmE660 strain without animal passage by introducing the same amino acid capsid substitutions. We evaluated one of the variants in rhesus macaques expressing permissive and restrictive TRIM5α alleles. The SIVsmE660 variant infected and replicated in macaques with restrictive TRIM5α genotypes as efficiently as in macaques with permissive TRIM5α genotypes. These results demonstrated that mutations in the SIV capsid can confer SIV resistance to TRIM5α restriction without animal passage, suggesting an applicable method to generate more diverse SIV strains for HIV vaccine studies. IMPORTANCE: Many strains of SIV from sooty mangabey monkeys are susceptible to resistance by common rhesus macaque TRIM5α alleles and result in reduced virus acquisition and replication in macaques that express these restrictive alleles. We previously observed that spontaneous variations in the capsid gene were associated with improved replication in macaques, and the introduction of two amino acid changes in the capsid transfers this improved replication to the parent clone. In the present study, we introduced these mutations into a related but distinct strain of SIV that is commonly used for challenge studies for vaccine trials. These mutations also improved the replication of this strain in macaques with the restrictive TRIM5α genotype and thus will eliminate the confounding effects of TRIM5α in vaccine studies.

Phylogeny and History of the Lost SIV from Crab-Eating Macaques: SIVmfa.

In the 20th century, thirteen distinct human immunodeficiency viruses emerged following independent cross-species transmission events involving simian immunodeficiency viruses (SIV) from African primates. In the late 1900s, pathogenic SIV strains also emerged in the United Sates among captive Asian macaque species following their unintentional infection with SIV from African sooty mangabeys (SIVsmm). Since their discovery in the 1980s, SIVs from rhesus macaques (SIVmac) and pig-tailed macaques (SIVmne) have become invaluable models for studying HIV pathogenesis, vaccine design and the emergence of viruses. SIV isolates from captive crab-eating macaques (SIVmfa) were initially described but lost prior to any detailed molecular and genetic characterization. In order to infer the origins of the lost SIVmfa lineage, we located archived material and colony records, recovered its genomic sequence by PCR, and assessed its phylogenetic relationship to other SIV strains. We conclude that SIVmfa is the product of two cross-species transmission events. The first was the established transmission of SIVsmm to rhesus macaques, which occurred at the California National Primate Research Center in the late 1960s and the virus later emerged as SIVmac. In a second event, SIVmac was transmitted to crab-eating macaques, likely at the Laboratory for Experimental Medicine and Surgery in Primates in the early 1970s, and it was later spread to the New England Primate Research Center colony in 1973 and eventually isolated in 1986. Our analysis suggests that SIVmac had already emerged by the early 1970s and had begun to diverge into distinct lineages. Furthermore, our findings suggest that pathogenic SIV strains may have been more widely distributed than previously appreciated, raising the possibility that additional isolates may await discovery.

Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

Evolutionary and Functional Analysis of Old World Primate TRIM5 Reveals the Ancient Emergence of Primate Lentiviruses and Convergent Evolution Targeting a Conserved Capsid Interface.

The widespread distribution of lentiviruses among African primates, and the lack of severe pathogenesis in many of these natural reservoirs, are taken as evidence for long-term co-evolution between the simian immunodeficiency viruses (SIVs) and their primate hosts. Evidence for positive selection acting on antiviral restriction factors is consistent with virus-host interactions spanning millions of years of primate evolution. However, many restriction mechanisms are not virus-specific, and selection cannot be unambiguously attributed to any one type of virus. We hypothesized that the restriction factor TRIM5, because of its unique specificity for retrovirus capsids, should accumulate adaptive changes in a virus-specific fashion, and therefore, that phylogenetic reconstruction of TRIM5 evolution in African primates should reveal selection by lentiviruses closely related to modern SIVs. We analyzed complete TRIM5 coding sequences of 22 Old World primates and identified a tightly-spaced cluster of branch-specific adaptions appearing in the Cercopithecinae lineage after divergence from the Colobinae around 16 million years ago. Functional assays of both extant TRIM5 orthologs and reconstructed ancestral TRIM5 proteins revealed that this cluster of adaptations in TRIM5 specifically resulted in the ability to restrict Cercopithecine lentiviruses, but had no effect (positive or negative) on restriction of other retroviruses, including lentiviruses of non-Cercopithecine primates. The correlation between lineage-specific adaptations and ability to restrict viruses endemic to the same hosts supports the hypothesis that lentiviruses closely related to modern SIVs were present in Africa and infecting the ancestors of Cercopithecine primates as far back as 16 million years ago, and provides insight into the evolution of TRIM5 specificity.

Monkey-adapted HIV-1 highlights in vivo significance of restriction factors.

HIV-1 was isolated 31 years ago, yet models for studying HIV-1 pathogenesis in vivo are still lacking. Recent experiments using an HIV-1 strain engineered to replicate in macaques recapitulate several important features of human AIDS, and provide insight into the genetics of cross-species transmission and emergence of pathogenic retroviruses.

Understanding restriction factors and intrinsic immunity: insights and lessons from the primate lentiviruses.

Primate lentiviruses include the HIVs, HIV-1 and HIV-2; the SIVs, which are endemic to more than 40 species of nonhuman primates in Africa; and SIVmac, an AIDS-causing pathogen that emerged in US macaque colonies in the 1970s. Because of the worldwide spread of HIV and AIDS, primate lentiviruses have been intensively investigated for more than 30 years. Research on these viruses has played a leading role in the discovery and characterization of intrinsic immunity, and in particular the identification of several antiviral effectors (also known as restriction factors) including APOBEC3G, TRIM5α, BST-2/tetherin and SAMHD1. Comparative studies of the primate lentiviruses and their hosts have proven critical for understanding both the evolutionary significance and biological relevance of intrinsic immunity, and the role intrinsic immunity plays in governing viral host range and interspecies transmission of viruses in nature.

TRIM5 alpha drives SIVsmm evolution in rhesus macaques.

The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5(Q) was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5(TFP/TFP) or TRIM5(Cyp/TFP). Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5(TFP) restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5(Cyp). Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIM(TFP) alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates.

Gain-of-sensitivity mutations in a Trim5-resistant primary isolate of pathogenic SIV identify two independent conserved determinants of Trim5α specificity.

Retroviral capsid recognition by Trim5 blocks productive infection. Rhesus macaques harbor three functionally distinct Trim5 alleles: Trim5α(Q) , Trim5α(TFP) and Trim5(CypA) . Despite the high degree of amino acid identity between Trim5α(Q) and Trim5α(TFP) alleles, the Q/TFP polymorphism results in the differential restriction of some primate lentiviruses, suggesting these alleles differ in how they engage these capsids. Simian immunodeficiency virus of rhesus macaques (SIVmac) evolved to resist all three alleles. Thus, SIVmac provides a unique opportunity to study a virus in the context of the Trim5 repertoire that drove its evolution in vivo. We exploited the evolved rhesus Trim5α resistance of this capsid to identify gain-of-sensitivity mutations that distinguish targets between the Trim5α(Q) and Trim5α(TFP) alleles. While both alleles recognize the capsid surface, Trim5α(Q) and Trim5α(TFP) alleles differed in their ability to restrict a panel of capsid chimeras and single amino acid substitutions. When mapped onto the structure of the SIVmac239 capsid N-terminal domain, single amino acid substitutions affecting both alleles mapped to the ß-hairpin. Given that none of the substitutions affected Trim5α(Q) alone, and the fact that the ß-hairpin is conserved among retroviral capsids, we propose that the ß-hairpin is a molecular pattern widely exploited by Trim5α proteins. Mutations specifically affecting rhesus Trim5α(TFP) (without affecting Trim5α(Q) ) surround a site of conservation unique to primate lentiviruses, overlapping the CPSF6 binding site. We believe targeting this site is an evolutionary innovation driven specifically by the emergence of primate lentiviruses in Africa during the last 12 million years. This modularity in targeting may be a general feature of Trim5 evolution, permitting different regions of the PRYSPRY domain to evolve independent interactions with capsid.

TRIM5 suppresses cross-species transmission of a primate immunodeficiency virus and selects for emergence of resistant variants in the new species.

Simian immunodeficiency viruses of sooty mangabeys (SIVsm) are the source of multiple, successful cross-species transmissions, having given rise to HIV-2 in humans, SIVmac in rhesus macaques, and SIVstm in stump-tailed macaques. Cellular assays and phylogenetic comparisons indirectly support a role for TRIM5alpha, the product of the TRIM5 gene, in suppressing interspecies transmission and emergence of retroviruses in nature. Here, we investigate the in vivo role of TRIM5 directly, focusing on transmission of primate immunodeficiency viruses between outbred primate hosts. Specifically, we retrospectively analyzed experimental cross-species transmission of SIVsm in two cohorts of rhesus macaques and found a significant effect of TRIM5 genotype on viral replication levels. The effect was especially pronounced in a cohort of animals infected with SIVsmE543-3, where TRIM5 genotype correlated with approximately 100-fold to 1,000-fold differences in viral replication levels. Surprisingly, transmission occurred even in individuals bearing restrictive TRIM5 genotypes, resulting in attenuation of replication rather than an outright block to infection. In cell-culture assays, the same TRIM5 alleles associated with viral suppression in vivo blocked infectivity of two SIVsm strains, but not the macaque-adapted strain SIVmac239. Adaptations appeared in the viral capsid in animals with restrictive TRIM5 genotypes, and similar adaptations coincide with emergence of SIVmac in captive macaques in the 1970s. Thus, host TRIM5 can suppress viral replication in vivo, exerting selective pressure during the initial stages of cross-species transmission.

Acquisition and processing of nonhuman primate samples for genetic and phylogenetic analyses.

Primates have long been a favorite subject of evolutionary biologists, and in recent decades, have come to play an increasingly important role in biomedical research, including comparative genetics and phylogenetics. The growing list of annotated genome databases from nonhuman primate species is expected to aid in these endeavors, allowing many analyses to be performed partially or even entirely in silico. However, whole genome sequence data are typically derived from only one, or at best a few, individuals. As a consequence, information in the databases does not capture variation within species or populations, nor can the sequence of one individual be taken as representative across all loci. Furthermore, the vast majority of primate species have not been sequenced, and only a small percentage of species are currently slated for whole genome sequencing efforts. Finally, for many species data on patterns and levels of RNA expression will be lacking. Thus, there will continue to be a demand for samples from nonhuman primates as raw material for genetic and phylogenetic analyses. Gathering such samples can be complicated, with many legal and practical barriers to obtaining samples in the field or transporting samples between research centers and across borders. Here, we provide basic but critical advice for those initiating studies requiring genetic material from nonhuman primates, including some guidance on how to locate and obtain samples, brief overviews of common protocols for handling and processing samples, and a table of useful links for locating resources related to the acquisition of samples. We also advocate for the creation of curated banks of nonhuman primate samples, particularly renewable sources of genetic material such as immortalized cell lines or fibroblasts, to reduce the need for repeated or redundant sampling from living animals.

Evolution of a TRIM5-CypA splice isoform in old world monkeys.

The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5-10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.