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Porcine Circovirus type (PCV) 2 is an important pathogen that has been circulating worldwide and has cuased serious economic loss in pig industry. However, both PCV3 and PCV4 are newly emerging viruses. In Taiwan, PCV2 has been one of the critical pathogens in pig frams and PCV3 has been detected since 2016; however, the epidemiolog of PCV3 in Taiwan remains unclear and PCV4 has yet to be identified. Therefore, in order to detect the positive rate of PCV2, to investigate the epidemiolog of PCV3 in the pig farms, and to examine whether pigs were infected with PCV4 in Taiwan, a total of 128 samples from 46 clinical cases of pigs were collected from September 2020 to December 2021. The case detection rates were 54.3 % for PCV2, 43.5 % for PCV3, and 2.2 % for PCV4. The results suggested that the positivity rates for both PCV2 and PCV3 were still high in Taiwan. In addition, PCV3 was detected among cases from all 7 sampled counties and in 11 of the 16 sampling months, suggesting that PCV3 may lead to endemic pig disease in Taiwan. Surprisingly, the PCV4 was also detected, suggesting the first PCV4 case in Taiwan. The complete genomes derived from the identified PCV3 and PCV4 strains were subsequently sequenced followed by phylogenetic analysis. The results suggested that the 17 identified PCV3 strains could be divided into Taiwanese-like and Japanese-like strains. In addition, the amino acid residues at positions 27, 80, and 212 in the identified PCV4 cap protein were asparagine, isoleucine, and methionine, respectively, and thus the identified PCV4 was catalorized into clade PCV4b. Consequently, it is concluded that (i) the prevalence of PCV2 and PCV3 is still high in Taiwanese pigs, (ii) PCV3 has may be an endemic infection in Taiwan and can be classified into Japanese-like and Taiwanese-like strains, (iii) PCV4 was detected for the first time in Taiwanese pigs and can be classified into PCV4b. It remains unclear how PCV2, PCV3, and PCV4 were introduced to Taiwan, and thus continuous investigation of emerging pathogens in pigs is needed.

Viruses in the family Retroviridae are found in a wide variety of vertebrate hosts. Enveloped virions are 80-100 nm in diameter with an inner core containing the viral genome and replicative enzymes. Core morphology is often characteristic for viruses within the same genus. Replication involves reverse transcription and integration into host cell DNA, resulting in a provirus. Integration into germline cells can result in a heritable provirus known as an endogenous retrovirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Retroviridae, which is available at ictv.global/report/retroviridae.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to suppress the type I interferon (IFNs-α/β) response during infection. PRRSV also activates the NF-κB signaling pathway, leading to the production of proinflammatory cytokines during infection. In swine farms, co-infections of PRRSV and other secondary bacterial pathogens are common and exacerbate the production of proinflammatory cytokines, contributing to the porcine respiratory disease complex (PRDC) which is clinically a severe disease. Previous studies identified the non-structural protein 1β (nsp1β) of PRRSV-2 as an IFN antagonist and the nucleocapsid (N) protein as the NF-κB activator. Further studies showed the leucine at position 126 (L126) of nsp1β as the essential residue for IFN suppression and the region spanning the nuclear localization signal (NLS) of N as the NF-κB activation domain. In the present study, we generated a double-mutant PRRSV-2 that contained the L126A mutation in the nsp1β gene and the NLS mutation (ΔNLS) in the N gene using reverse genetics. The immunological phenotype of this mutant PRRSV-2 was examined in porcine alveolar macrophages (PAMs) in vitro and in young pigs in vivo. In PAMs, the double-mutant virus did not suppress IFN-β expression but decreased the NF-κB-dependent inflammatory cytokine productions compared to those for wild-type PRRSV-2. Co-infection of PAMs with the mutant PRRSV-2 and Streptococcus suis (S. suis) also reduced the production of NF-κB-directed inflammatory cytokines. To further examine the cytokine profiles and the disease severity by the mutant virus in natural host animals, 6 groups of pigs, 7 animals per group, were used for co-infection with the mutant PRRSV-2 and S. suis. The double-mutant PRRSV-2 was clinically attenuated, and the expressions of proinflammatory cytokines and chemokines were significantly reduced in pigs after bacterial co-infection. Compared to the wild-type PRRSV-2 and S. suis co-infection control, pigs coinfected with the double-mutant PRRSV-2 exhibited milder clinical signs, lower titers and shorter duration of viremia, and lower expression of proinflammatory cytokines. In conclusion, our study demonstrates that genetic modification of the type I IFN suppression and NF-κB activation functions of PRRSV-2 may allow us to design a novel vaccine candidate to alleviate the clinical severity of PRRS-2 and PRDC during bacterial co-infection.

Tripartite motif (TRIM)-containing proteins are a family of regulatory proteins that can participate in the induction of antiviral cytokines and antagonize viral replication. Promyelocytic leukemia (PML) protein is known as TRIM19 and is a major scaffold protein organizing the PML nuclear bodies (NBs). PML NBs are membrane-less organelles in the nucleus and play a diverse role in maintaining cellular homeostasis including antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV), a member virus of the family Arteriviridae, inhibits type I interferon (IFN) response during infection, and nonstructural protein 1 (nsp1) of the virus has been identified as a potent IFN antagonist. We report that the numbers of PML NBs per nucleus were significantly downregulated during infection of PRRSV. The overexpression of all six isoforms of PML suppressed the PRRSV replication, and conversely, the silencing of PML gene expression enhanced the PRRSV replication. The suppression of PML NBs by the nsp1 protein was common in other member viruses of the family, represented by equine arteritis virus, lactate dehydrogenase elevating virus of mice, and simian hemorrhagic fever virus. Our study unveils a conserved viral strategy in arteriviruses for innate immune evasion.

Between January 2015 and October 2022, 38 patients with culture-confirmed melioidosis were identified in the Kowloon West (KW) Region, Hong Kong. Notably, 30 of them were clustered in the Sham Shui Po (SSP) district, which covers an estimated area of 2.5 km2. Between August and October 2022, 18 patients were identified in this district after heavy rainfall and typhoons. The sudden upsurge in cases prompted an environmental investigation, which involved collecting 20 air samples and 72 soil samples from residential areas near the patients. A viable isolate of Burkholderia pseudomallei was obtained from an air sample collected at a building site five days after a typhoon. B. pseudomallei DNA was also detected in 21 soil samples collected from the building site and adjacent gardening areas using full-length 16S rRNA gene sequencing, suggesting that B. psuedomallei is widely distributed in the soil environment surrounding the district. Core genome-multilocus sequence typing showed that the air sample isolate was phylogenetically clustered with the outbreak isolates in KW Region. Multispectral satellite imagery revealed a continuous reduction in vegetation region in SSP district by 162,255 m2 from 2016 to 2022, supporting the hypothesis of inhalation of aerosols from the contaminated soil as the transmission route of melioidosis during extreme weather events. This is because the bacteria in unvegetated soil are more easily spread by winds. In consistent with inhalational melioidosis, 24 (63.2%) patients had pneumonia. Clinicians should be aware of melioidosis during typhoon season and initiate appropriate investigation and treatment for patients with compatible symptoms.

Retroviral integration into germline DNA can result in the formation of a vertically inherited proviral sequence called an endogenous retrovirus (ERV). Over the course of their evolution, vertebrate genomes have accumulated many thousands of ERV loci. These sequences provide useful retrospective information about ancient retroviruses, and have also played an important role in shaping the evolution of vertebrate genomes. There is an immediate need for a unified system of nomenclature for ERV loci, not only to assist genome annotation, but also to facilitate research on ERVs and their impact on genome biology and evolution. In this review, we examine how ERV nomenclatures have developed, and consider the possibilities for the implementation of a systematic approach for naming ERV loci. We propose that such a nomenclature should not only provide unique identifiers for individual loci, but also denote orthologous relationships between ERVs in different species. In addition, we propose that-where possible-mnemonic links to previous, well-established names for ERV loci and groups should be retained. We show how this approach can be applied and integrated into existing taxonomic and nomenclature schemes for retroviruses, ERVs and transposable elements.

Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.

Advancing knowledge of biological mechanisms has come to depend upon genetic manipulation of cells and organisms, relying upon cellular cloning methods that remain unchanged for decades, are labor and time intensive, often taking many months to come to fruition. Thus, there is a pressing need for more efficient processes. We have adapted a newly developed micropallet array platform, termed the "ferro-core micropallet array", to dramatically improve and accelerate the process of isolating clonal populations of adherent cells from heterogeneous mixtures retaining the flexibility of employing a wide range of cytometric parameters for identifying colonies and cells of interest. Using transfected (retroviral oncogene or fluorescent reporter construct) rat 208 F cells, we demonstrated the capacity to isolate and expand pure populations of genetically manipulated cells via laser release and magnetic recovery of single micropallets carrying adherent microcolonies derived from single cells. This platform can be broadly applied to biological research, across the spectrum of molecular biology to cellular biology, involving fields such as cancer, developmental, and stem cell biology. The ferro-core micropallet array platform provides significant advantages over alternative sorting and cloning methods by eliminating the necessity for repetitive purification steps and increasing throughput by dramatically shortening the time to obtain clonally expanded cell colonies.

While virus growth dynamics have been well-characterized in several infections, data are typically collected once the virus population becomes easily detectable. Earlier dynamics, however, remain less understood. We recently reported unusual early dynamics in an experimental system using adenovirus infection of human embryonic kidney (293) cells. Under identical experimental conditions, inoculation at low infection multiplicities resulted in either robust spread, or in limited spread that eventually stalled, with both outcomes occurring with approximately equal frequencies. The reasons underlying these observations have not been understood. Here, we present further experimental data showing that inhibition of interferon-induced antiviral states in cells results in a significant increase in the percentage of robust infections that are observed, implicating a race between virus replication and the spread of the anti-viral state as a central mechanism. Analysis of a variety of computational models, however, reveals that this alone cannot explain the simultaneous occurrence of both viral growth outcomes under identical conditions, and that additional biological mechanisms have to be invoked to explain the data. One such mechanism is the ability of the virus to overcome the antiviral state through multiple infection of cells. If this is included in the model, two outcomes of viral spread are found to be simultaneously stable, depending on initial conditions. In stochastic versions of such models, the system can go by chance to either state from identical initial conditions, with the relative frequency of the outcomes depending on the strength of the interferon-based anti-viral response, consistent with the experiments. This demonstrates considerable complexity during the early phase of the infection that can influence the ability of a virus to become successfully established. Implications for the initial dynamics of oncolytic virus spread through tumors are discussed.

BackgroundKoala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag.ResultsIn this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results.ConclusionsThese results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction.

UnlabelledThe native envelope gene (env) of Jaagsiekte sheep retrovirus (JSRV) also acts as an oncogene. To investigate the mechanism of transformation, we performed yeast 2-hybrid screening for cellular proteins that interact with Env. Among several candidates, we identified mouse or rat zinc finger protein 111 (zfp111). The interaction between Env and Zfp111 was confirmed through in vivo coimmunoprecipitation assays. Knockdown of endogenous Zfp111 caused a decrease in cell transformation by JSRV Env, while overexpression of Zfp111 increased overall Env transformation, supporting a role for Zfp111 in Env transformation. Knockdown of Zfp111 had no effect on the growth rate of parental rat 208F cells, while it decreased the proliferation rate of JSRV-transformed 208F cells, suggesting that JSRV-transformed cells became dependent on Zfp111. In addition, Zfp111 preferentially bound to a higher-mobility form of JSRV Env that has not been described previously. The higher-mobility form of Env (P70(env)) was found exclusively in the nuclear fraction, and size of its polypeptide backbone was the same as that of the cytoplasmic Env polyprotein (Pr80(env)). The differences in glycosylation between the two versions of Env were characterized. These results identify a novel cellular protein, Zfp111, that binds to the JSRV Env protein, and this binding plays a role in Env transformation. These results indicate that JSRV transformation also involves proteins and interactions in the nucleus.ImportanceThe envelope protein (Env) of Jaagsiekte sheep retrovirus (JSRV) is an oncogene, but its mechanism of cell transformation is still unclear. Here we identified seven candidate cellular proteins that can interact with JSRV Env by yeast two-hybrid screening. This study focused on one of the seven candidates, zinc finger protein 111 (Zfp111). Zfp111 was shown to interact with JSRV Env in cells and to be involved in JSRV transformation. Moreover, coexpression of JSRV Env and Zfp111 led to the identification of a novel nuclear form of the JSRV Env protein that binds Zfp111. Nuclear Env was found to differ by glycosylation from the cytoplasmic Env precursor to the virion envelope proteins. These results suggest that JSRV Env transformation may involve nuclear events such as an alteration in transcription mediated by Env-Zfp111 interactions.

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in 2006 in a search for a viral etiology of human prostate cancer (PC). Substantial interest in XMRV as a potentially new pathogenic human retrovirus was driven by reports that XMRV could be detected in a significant percentage of PC samples, and also in tissues from patients with chronic fatigue syndrome (CFS). After considerable controversy, etiologic links between XMRV and these two diseases were disproven. XMRV was determined to have arisen during passage of a human PC tumor in immunocompromised nude mice, by activation and recombination between two endogenous murine leukemia viruses from cells of the mouse. The resulting XMRV had a xentropic host range, which allowed it replicate in the human tumor cells in the xenograft. This review describes the discovery of XMRV, and the molecular and virological events leading to its formation, XMRV infection in animal models and biological effects on infected cells. Lessons from XMRV for other searches of viral etiologies of cancer are discussed, as well as cautions for researchers working on human tumors or cell lines that have been passed through nude mice, includingpotential biohazards associated with XMRV or other similar xenotropic murine leukemia viruses (MLVs).

Abstract Background ZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010). Methods To determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies. Results Mice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection. Conclusions These studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection.

AbstractBackgroundOne of the unique features of gammaretroviruses is that they contain an additional extended form of Gag, glyco-gag, which initiates in the leader sequence. MuLV glyco-gag, gPr80Gag, promotes retrovirus replication and disease progression. Although virtually all infectious MuLVs encode glyco-gag, XMRV (xenotropic murine leukemia virus-related virus) lacks the classical gPr80Gag sequence. We examined XMRV to determine if its leader sequence contains glyco-gag activity, whether the presence of conventional gPr80Gag affects replication of XMRV, and we describe the evolution of glyco-gag-deficient MuLVs in Mus.ResultsWe introduced several mutations disrupting two putative but noncanonical glyco-gag proteins in the leader sequence region in XMRV and found that those mutations did not affect virus release nor susceptibility to the antiviral activity of hA3G (human APOBEC3G). A chimeric XMRV encoding the Moloney MuLV (M-MuLV) leader sequence (MXMRV) demonstrated that M-MuLV glyco-gag facilitated MXMRV release and increased infectivity. Infectivity assays with several cell lines showed that glyco-gag increases XMRV infectivity in all cell lines tested, but the level of this increase varies in different cell lines. Because MuLV glyco-gag counteracts mouse APOBEC3, we investigated whether M-MuLV glyco-gag enhances XMRV infection by counteracting human APOBEC3. Comparison of hAPOBEC3 isoforms expressed in different cell lines indicated that hA3B was the most likely candidate for a restrictive hA3. However over-expression of hA3B showed no enhanced restriction of infection by XMRV compared to MXMRV. Endogenous MuLVs in the sequenced mouse genome were screened for canonical glyco-gag, which was identified in two clades of xenotropic MuLVs (X-MuLVs) and ecotropic MuLVs, but not in other X-MuLVs or in any polytropic MuLVs.ConclusionsM-MuLV glyco-gag facilitates XMRV replication, and the leader sequence region in XMRV does not encode proteins equivalent to M-MuLV glyco-gag. The fact that the ability of glyco-gag to enhance XMRV infection varies in different cell lines suggests a glyco-gag sensitive restrictive factor that further reduces XMRV infectivity. The M-MuLV glyco-gag enhancement for XMRV replication is through a hAPOBEC3 independent mechanism. The absence of glyco-gag in MuLVs carried by western European mice suggests that loss of this sequence is a relatively recent event with limited subspecies distribution.

Studies of oncogenic viruses have made seminal contributions to the molecular biology of cancer. Key discoveries include the identification of viral oncogenes and cellular proto-oncogenes, elucidation of signal transduction pathways, and identification of tumor suppressor genes. The origins of cancer virology began almost exactly one hundred years ago with the discovery of avian sarcoma and acute leukemia viruses—RNA-containing viruses of the retrovirus family. The study of animal cancer viruses accelerated beginning in the late 1960s and early 1970s, with the discovery of DNA viruses that could transform cells in culture, and the development of quantitative assays for transformation by DNA and RNA-containing tumor viruses. The discovery of reverse transcriptase in retroviruses in 1970 also greatly accelerated research on these viruses. Indeed RNA and DNA tumor viruses led the way in cancer molecular biology during this era before molecular cloning. It was possible to physically purify virus particles and generate specific hybridization probes for viral DNA and RNA at a time when it was not possible to analyze cellular genes in the same manner.

Retroviruses cause cancers in a variety of animals and humans. Research on retroviruses has provided important insights into mechanisms of oncogenesis in humans, including the discovery of viral oncogenes and cellular proto-oncogenes. The subject of this review is the mechanisms by which retroviruses that do not carry oncogenes (non-acute retroviruses) cause cancers. The common theme is that these tumors result from insertional activation of cellular proto-oncogenes by integration of viral DNA. Early research on insertional activation of proto-oncogenes in virus-induced tumors is reviewed. Research on non-acute retroviruses has led to the discovery of new proto-oncogenes through searches for common insertion sites (CISs) in virus-induced tumors. Cooperation between different proto-oncogenes in development of tumors has been elucidated through the study of retrovirus-induced tumors, and retroviral infection of genetically susceptible mice (retroviral tagging) has been used to identify cellular proto-oncogenes active in specific oncogenic pathways. The pace of proto-oncogene discovery has been accelerated by technical advances including PCR cloning of viral integration sites, the availability of the mouse genome sequence, and high throughput DNA sequencing. Insertional activation has proven to be a significant risk in gene therapy trials to correct genetic defects with retroviral vectors. Studies on non-acute retroviral oncogenesis provide insight into the potential risks, and the mechanisms of oncogenesis.

Murine leukemia viruses (MuLVs) encode two forms of Gag polyprotein: the precursor for the viral core proteins (Pr65(gag) for Moloney MuLV [M-MuLV]) and a longer glycosylated form (glyco-gag, or gPr80(gag)). gPr80gag is translated from the same unspliced viral RNA as Pr65(gag), from an upstream in-frame CUG initiation codon. As a result, gPr80(gag) contains 88 unique N-terminal amino acids that include a signal peptide that conducts gPr80(gag) into the rough endoplasmic reticulum, where it is glycosylated, exported to the cell surface, and cleaved into two proteins of 55 and 40 kDa. The amino-terminal 55-kDa protein remains cell associated with the 88 unique amino acids exposed to the cytosol. We previously showed that gPr80(gag) facilitates efficient M-MuLV release through lipid rafts. In this report, we found that the unique N-terminal domain of gPr80(gag) is sufficient to facilitate enhanced M-MuLV particle release from transfected 293T cells. A search for cellular proteins involved in gPr80(gag) function led to cellular La protein. Overexpression of mouse or human La enhanced M-MuLV particle release in the absence of glyco-gag, and the released virus had a reduced buoyant density characteristic of increased cholesterol content. Moreover, small interfering RNA (siRNA) knockdown of human La abolished glyco-gag enhancement of M-MuLV release. These results implicate La as a cellular protein involved in M-MuLV glyco-gag function. We also found that overexpression of mouse or human La could enhance HIV-1 release in the absence of gPr80(gag). Therefore, M-MuLV and HIV-1 may share a pathway for release through lipid rafts involving La. IMPORTANCE Retroviruses cause diseases such as leukemia and AIDS. An important aspect of viral replication is how viruses are released from infected cells. We previously found that a unique protein encoded by murine leukemia viruses (MuLVs), glyco-gag (or gPr80(gag)), enhances efficient virus release through cholesterol-rich membrane subdomains called lipid rafts. In this study, we found that the N-terminal domain of gPr80(gag) is sufficient to enhance viral release. A search for cellular proteins that participate in gPr80(gag) function led to cellular La protein. Overexpression of La phenocopied glyco-gag in enhancing M-MuLV release, and knockdown of La abolished glyco-gag function. M-MuLV glyco-gag also enhanced release of HIV-1, as did overexpression La in the absence of glyco-gag. Thus, M-MuLV and HIV-1 may share a cellular pathway for release through lipid rafts involving La. These results may also be relevant for other viruses that are released through lipid rafts.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology.

Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65(gag), the precursor for internal structural (Gag) proteins. Whereas gPr80gag is evolutionarily conserved among gammaretroviruses, its mechanism of action has been unclear, although it facilitates virus production at a late assembly or release step. Here, it is shown that gPr80gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular, gPr80gag-facilitated release occurs through lipid rafts, because gPr80gag-negative M-MuLV has a lower cholesterol content, is less sensitive to inhibition of release by the cholesterol-depleting agent MbetaCD, and there is less Pr65gag associated with detergent-resistant membranes in mutant-infected cells. gPr80gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.

All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80gag) in addition to the polyprotein precursor of the viral capsid proteins (Pr65gag). gPr80gag is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65gag. The role of glyco-Gag in MuLV replication has been unclear, since gPr80gag-negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80gag mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80gag negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80gag in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80gag produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80gag is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.

Background: Dynamic susceptibility contrast (DSC) MR perfusion is a frequently-used technique for neurovascular imaging. The progress of a bolus of contrast agent through the tissue of the brain is imaged via a series of T2*-weighted MRI scans. Clinically relevant parameters such as blood flow and Tmax can be calculated by deconvolving the contrast-time curves with the bolus shape (arterial input function). In acute stroke, for instance, these parameters may help distinguish between the likely salvageable tissue and irreversibly damaged infarct core. Deconvolution typically relies on singular value decomposition (SVD): however, studies have shown that these algorithms are very sensitive to noise and artifacts present in the image and therefore may introduce distortions that influence the estimated output parameters.Methods: In this work, we present a machine learning approach to the estimation of perfusion parameters in DSC-MRI. Various machine learning models using as input the raw MR source data were trained to reproduce the output of an FDA approved commercial implementation of the SVD deconvolution algorithm. Experiments were conducted to determine the effect of training set size, optimal patch size, and the effect of using different machine-learning models for regression.Results: Model performance increased with training set size, but after 5,000 samples (voxels) this effect was minimal. Models inferring perfusion maps from a 5 by 5 voxel patch outperformed models able to use the information in a single voxel, but larger patches led to worse performance. Random Forest models produced had the lowest root mean squared error, with neural networks performing second best: however, a phantom study revealed that the random forest was highly susceptible to noise levels, while the neural network was more robust.Conclusion: The machine learning-based approach produces estimates of the perfusion parameters invariant to the noise and artifacts that commonly occur as part of MR acquisition. As a result, better robustness to noise is obtained, when evaluated against the FDA approved software on acute stroke patients and simulated phantom data.