Is the Gut the Major Source of Virus in Early Simian Immunodeficiency Virus Infection?

The acute phases of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection are characterized by rapid and profound depletion of CD4⁺ T cells from the guts of infected individuals. The large number of CD4⁺ T cells in the gut (a large fraction of which are activated and express the HIV/SIV coreceptor CCR5), the high level of infection of these cells, and the temporal coincidence of this CD4⁺ T-cell depletion with the peak of virus in plasma in acute infection suggest that the intestinal mucosa may be the major source of virus driving the peak viral load. Here, we used data on CD4⁺ T-cell proportions in the lamina propria of the rectums of SIV-infected rhesus macaques (which progress to AIDS) and sooty mangabeys (which do not progress) to show that in both species, the depletion of CD4⁺ T cells from this mucosal site and its maximum loss rate are often observed several days before the peak in viral load, with few CD4⁺ T cells remaining in the rectum by the time of peak viral load. In contrast, the maximum loss rate of CD4⁺ T cells from bronchoalveolar lavage specimens and lymph nodes coincides with the peak in virus. Analysis of the kinetics of depletion suggests that, in both rhesus macaques and sooty mangabeys, CD4⁺ T cells in the intestinal mucosa are a highly susceptible population for infection but not a major source of plasma virus in acute SIV infection.The acute phase of human immunodeficiency virus (HIV) infection is characterized by moderate CD4⁺ T-cell depletion in blood, followed by a transient partial restoration of CD4⁺ T-cell numbers and eventually by a slow long-term CD4⁺ T-cell decline in the chronic phase that lasts for several years. Studies of CD4⁺ T-cell depletion in mucosal sites, often conducted with simian immunodeficiency virus (SIV)-infected macaques, have demonstrated that mucosal CD4⁺ T-cell depletion is more rapid and profound (3, 10, 13, 19, 21). The severe depletion of cells in the gut in early infection is thought to be driven in part by the phenotype of the cells present, which are predominantly CCR5⁺ and in general more activated than their circulating counterparts. As such, these mucosal CD4⁺ T cells are highly susceptible to productive infection with the dominant CCR5-tropic strains of HIV and SIV present in early infection (20). The rapid depletion of CD4⁺ T cells at mucosal sites is accompanied by relatively high numbers of infected cells (10, 13) and is temporally associated with the peak viral load in plasma, suggesting that the infection of mucosal CD4⁺ T cells may be responsible for the majority of virus replication occurring during acute infection (10, 15, 21, 22).The size of the CD4⁺ T-cell pool in the gut is a matter of some controversy, with estimates ranging from ∼5 to 50% of the total body pool of these cells (reviewed in reference 5). Regardless of the precise numbers, the gut (and particularly the mucosal lamina propria) contains a significant proportion of the body CD4⁺ CCR5⁺ memory T cells, which are depleted very early in infection. However, whether CD4⁺ T cells in the gut are merely a target of early infection or whether they are a major driver of early viral growth and peak viral loads in acute infection is unclear. Here we use a combination of experimental data and modeling to demonstrate that the gut is unlikely to be a major source of virus production in acute SIV infection.     

14. Studies on Poliomyelitis Virus—Concentration and Purification of the Virus

ABSTRACT

For the purpose of determining the immunogenic potency of polio virus, relatively large amounts of concentrated virus material were prepared which had titres of the order of 10¹⁰ T.C.I.D.jo per ml. These were obtained by pervaporating large quantities of tissue culture fluid containing approximately 10⁶⁵ T.C.I.D.JQ per ml.     

Dissecting Virus Entry: Replication-Independent Analysis of Virus Binding,Internalization, and Penetration Using Minimal Complementation of β-Galactosidase

Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).     

The Advantage of Sex in the RNA Virus φ6

When laboratory populations of the RNA bacteriophage 6 are subjected to intensified genetic drift, they experience a decline in fitness. These experiments demonstrate that the average effect of mutations is deleterious, and they are used to suggest that Muller's ratchet can operate in these viruses. However, the operation of Muller's ratchet does not alone guarantee an advantage of sex. When 6 populations were subjected to a series of bottlenecks of one individual and then crossed, the measured advantage of sex was not significant. To determine whether a small sample size, as opposed to allelism or another explanation, can account for the negative result, we repeated the 6 experiments by crossing a larger set of populations. We found that bottlenecked populations of 6 could recover fitness through mutations. However, hybrids produced by crossing the populations recovered an additional amount over the contribution of mutations. This additional amount, which represents an advantage of sex to 6, was determined to be significantly greater than zero. These results provide indirect support for an advantage of sex through Muller's ratchet. However, we also use our experimental design and results to propose an alternative to Muller's ratchet as a model for the evolution of sex.     

Venezuelan Equine Encephalitis Virus Activity in the Gulf Coast Region of Mexico, 2003–2010

Venezuelan equine encephalitis virus (VEEV) has been the causative agent for sporadic epidemics and equine epizootics throughout the Americas since the 1930s. In 1969, an outbreak of Venezuelan equine encephalitis (VEE) spread rapidly from Guatemala and through the Gulf Coast region of Mexico, reaching Texas in 1971. Since this outbreak, there have been very few studies to determine the northward extent of endemic VEEV in this region. This study reports the findings of serologic surveillance in the Gulf Coast region of Mexico from 2003–2010. Phylogenetic analysis was also performed on viral isolates from this region to determine whether there have been substantial genetic changes in VEEV since the 1960s. Based on the findings of this study, the Gulf Coast lineage of subtype IE VEEV continues to actively circulate in this region of Mexico and appears to be responsible for infection of humans and animals throughout this region, including the northern State of Tamaulipas, which borders Texas.     

Expression of the Cloned Hemagglutinin–Neuraminidase Gene of the Newcastle Disease Virus in Mouse Myeloma Cells

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin–neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoic fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.     

High Prevalence of Hepatitis E Virus in Swedish Moose – A Phylogenetic Characterization and Comparison of the Virus from Different Regions

Background

Hepatitis E virus (HEV) infects a range of species, including humans, pigs, wild boars and deer. Zoonotic transmission may contribute to the high HEV seroprevalence in the human population of many countries. A novel divergent HEV from moose (Alces alces) in Sweden was recently identified by partial genome sequencing. Since only one strain was found, its classification within the HEV family, prevalence in moose and zoonotic potential was unclear. We therefore investigated samples from 231 moose in seven Swedish counties for HEV, and sequenced a near complete moose HEV genome. Phylogenetic analysis to classify this virus within the family Hepeviridae and to explore potential host specific determinants was performed.

Methods and Findings

The HEV prevalence of moose was determined by PCR (marker for active infection) and serological assays (marker of past infection) of sera and 51 fecal samples from 231 Swedish moose. Markers of active and past infection were found in 67 (29%) animals, while 34 (15%) were positive for HEV RNA, 43 (19%) were seropositive for anti-HEV antibodies, and 10 (4%) had both markers. The number of young individuals positive for HEV RNA was larger than for older individuals, and the number of anti-HEV antibody positive individuals increased with age. The high throughput sequenced moose HEV genome was 35-60% identical to existing HEVs. Partial ORF1 sequences from 13 moose strains showed high similarity among them, forming a distinct monophyletic clade with a common ancestor to HEV genotype 1-6 group, which includes members known for zoonotic transmission.

Conclusions

This study demonstrates a high frequency of HEV in moose in Sweden, with markers of current and past infection demonstrated in 30% of the animals. Moose is thus an important animal reservoir of HEV. The phylogenetic relationship demonstrated that the moose HEV belonged to the genotype 1-6 group, which includes strains that also infect humans, and therefore may signify a potential for zoonotic transmission of this HEV.     

Purification of the α Subunit of Avian Myeloblastosis Virus DNA Polymerase by Polyuridylic Acid-Sepharose

The alpha subunit of the avian myeloblastosis virus DNA polymerase could be readily purified to near homogeneity using a polyuridylic acid-Sepharose column chromatography step.     

Molecular Characterization of Human Pathogenic Bunyaviruses of the Nyando and Bwamba/Pongola Virus Groups Leads to the Genetic Identification of Mojuí dos Campos and Kaeng Khoi Virus

Background

Human infection with Bwamba virus (BWAV) and the closely related Pongola virus (PGAV), as well as Nyando virus (NDV), are important causes of febrile illness in Africa. However, despite seroprevalence studies that indicate high rates of infection in many countries, these viruses remain relatively unknown and unstudied. In addition, a number of unclassified bunyaviruses have been isolated over the years often with uncertain relationships to human disease.

Methodology/Principal Findings

In order to better understand the genetic and evolutionary relationships among orthobunyaviruses associated with human disease, we have sequenced the complete genomes for all 3 segments of multiple strains of BWAV (n = 2), PGAV (n = 2) and NDV (n = 4), as well as the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi viruses (KKV). Based on phylogenetic analysis, we show that these viruses populate 2 distinct branches, one made up of BWAV and PGAV and the other composed of NDV, MDCV and KKV. Interestingly, the NDV strains analyzed form two distinct clades which differed by >10% on the amino acid level across all protein products. In addition, the assignment of two bat-associated bunyaviruses into the NDV group, which is clearly associated with mosquito-borne infection, led us to analyze the ability of these different viruses to grow in bat (RE05 and Tb 1 Lu) and mosquito (C6/36) cell lines, and indeed all the viruses tested were capable of efficient growth in these cell types.

Conclusions/Significance

On the basis of our analyses, it is proposed to reclassify the NDV strains ERET147 and YM176-66 as a new virus species. Further, our analysis definitively identifies the previously unclassified bunyaviruses MDCV and KKV as distinct species within the NDV group and suggests that these viruses may have a broader host range than is currently appreciated.     

Antigenic and Morphological Similarities of Progressive Pneumonia Virus, a Recently Isolated “Slow Virus” of Sheep, to Visna and Maedi Viruses

Progressive pneumonia virus, the causative agent of a slow, pulmonary disease of Montana sheep, was shown to be antigenically related to two other slow viruses of sheep, visna and maedi. Electron microscopic examination of infected cells revealed that the virus matures by a budding process and that the budding particles as well as the mature, extracellular virions bear striking resemblances to the oncogenic ribonucleic acid (RNA) viruses. Recent findings of an RNA-dependent deoxyribonucleic acid polymerase associated with the virions of this group of slow viruses lend further support to the notion that they may tentatively be classified with the oncogenic RNA tumor viruses.     

Transformation of Murine Cells by Two “Slow Viruses,” Visna Virus and Progressive Pneumonia Virus

Visna and progressive pneumonia virus (PPV), two antigenically related, non-oncogenic "slow viruses" which have ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, were examined for their ability to transform cells. Murine cells which had been exposed to either visna or PPV developed foci of altered, spindle-shaped cells 3 to 4 weeks after infection. Visna and PPV transformed lines were established from these cultures. There was no evidence that other oncogenic DNA or RNA viruses were involved in the observed transformation. Visna or PPV could be "rescued" from all transformed lines by co-cultivation with normal sheep testis cells. "Rescued" virus was identified as visna or PPV, and they retained the capacity to transform mouse cells. These experiments may have important implications in the understanding of both viral carcinogenesis and "slow" viral infections.     

Analysis of Influence of Baicalin Joint Resveratrol Retention Enema on the TNF-α, SIgA,IL-2, IFN-γ of Rats with Respiratory Syncytial Virus Infection

Explore the influence of baicalin joint resveratrol retention enema on TNF-α, SIgA, IL-2, and IFN-γ of rats with respiratory syncytial virus (RSV) infection. The 60 SD rats were randomly divided into normal group, model group, baicalin group, resveratrol group, joint group, and ribavirin group. For model group, baicalin group, resveratrol group, joint group, and ribavirin group, rats were given RSV virus suspension intranasally for 3 days, and model group was not given administration. Baicalin group, resveratrol group, joint group, and ribavirin group were, respectively, given baicalin 100 mg/kg/day, resveratrol 30 mg/kg/day, baicalin joint resveratrol, and ribavirin 1 g/kg/day retention enema. After continuously given administration 7 days, rats were measured in serum TNF-α, IL-2, IFN-γ levels and SIgA levels in bronchoalveolar lavage fluid. Model group, TNF-α, IL-2, IFN-γ, and SIgA were significantly higher than the normal group (P < 0.05); Baicalin group, resveratrol group, ribavirin group, TNF-α, IL-2, IFN-γ, and SIgA were significantly higher than the model group (P < 0.05); TNF-α, IL-2 between baicalin group, resveratrol group, ribavirin group, have no significant difference (P > 0.05); Baicalin group, resveratrol group, joint group, IFN-γ, and SIgA were significantly higher than the ribavirin group (P < 0.05); Joint group TNF-α, IL-2, IFN-γ, and SIgA were significantly higher than baicalin group, resveratrol group, and ribavirin group (P < 0.05). Baicalin joint resveratrol retention enema can increase RSV infection model in rats serum TNF-α, IL-2, IFN-γ levels and SIgA levels in bronchoalveolar lavage fluid, which may anti-virus through this mechanism.     

Structural and Functional Elements of the Promoter Encoded by the 5′ Untranslated Region of the Venezuelan Equine Encephalitis Virus Genome

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. The pathogenesis of this virus depends strongly on the sequences of the structural proteins and on the mutations in the RNA promoter encoded by the 5′ untranslated region (5′UTR) of the viral genome. In this study, we performed a detailed investigation of the structural and functional elements of the 5′-terminal promoter and analyzed the effect of multiple mutations introduced into the VEEV 5′UTR on virus and RNA replication. The results of this study demonstrate that RNA replication is determined by two synergistically functioning RNA elements. One of them is a very 5′-terminal AU dinucleotide, which is not involved in the stable RNA secondary structure, and the second is a short, G-C-rich RNA stem. An increase or decrease in the stem''s stability has deleterious effects on virus and RNA replication. In response to mutations in these RNA elements, VEEV replicative machinery was capable of developing new, compensatory sequences in the 5′UTR either containing 5′-terminal AUG or AU repeats or leading to the formation of new, heterologous stem-loops. Analysis of the numerous compensatory mutations suggested that at least two different mechanisms are involved in their generation. Some of the modifications introduced into the 5′ terminus of the viral genome led to an accumulation of the mutations in the VEEV nsPs, which suggested to us that there is a direct involvement of these proteins in promoter recognition. Furthermore, our data provide new evidence that the 3′ terminus of the negative-strand viral genome in the double-stranded RNA replicative intermediate is represented by a single-stranded RNA. Both the overall folding and the sequence determine its efficient function as a promoter for VEEV positive-strand RNA genome synthesis.Alphaviruses are a group of important human and animal pathogens. They are widely distributed both in the New and the Old Worlds and circulate between mosquito vectors and vertebrate hosts (45). In mosquitoes, they cause a persistent, life-long infection characterized by virus accumulation in salivary glands, which is required for infecting vertebrate hosts during a blood meal (50). In vertebrates, alphaviruses develop high-titer viremia, and their replication induces a variety of diseases with symptoms depending on both the host and the causative virus (11). Venezuelan equine encephalitis virus (VEEV), the New World alphavirus, is one of the most pathogenic members of the genus (16, 45). Representatives of the VEEV serocomplex circulate in Central, South, and North America and cause severe, and sometimes fatal, encephalitis in humans and horses (3, 11, 16, 24). Accordingly, VEEV represents a serious public health threat in the United States (39, 48, 51, 53), and during VEEV epizootics, equine mortality can reach 83%, and in humans, neurological diseases can be detected in up to 14% of all infected individuals, especially children (15). The overall mortality rate for humans is below 1%, but it is usually higher among children, the elderly, and, most likely, immunocompromised individuals (49). In spite of the continuous threat of VEEV epidemics, the biology of this virus, its pathogenesis, and the mechanism of replication are insufficiently understood. To date, no safe and efficient vaccine and therapeutic means have been developed for this pathogen.The VEEV genome is represented by a single-stranded, almost 11.5-kb-long RNA molecule of positive polarity. This RNA mimics the structure of cellular mRNAs by containing a cap at the 5′ ends and a poly(A) tail at the 3′ ends of the genome (18). The genomic RNA encodes two polyproteins: the 5′-terminal open reading frame (ORF) is translated into viral nonstructural proteins (nsP1 to nsP4), forming the replication enzyme complex (RC). The second ORF corresponds to the 3′-terminal one-third of the genome and encodes all of the viral structural proteins, C, E2, and E1. The latter proteins are translated from the subgenomic RNA synthesized during virus replication (45).The replication of the alphavirus genome is a highly regulated, multistep process, which includes the synthesis of three different RNA species (45). The regulation of their synthesis is achieved by differential processing of viral nsPs (22, 23, 43). First, the initially synthesized nonstructural polyprotein is partially processed by the nsP2-associated protease into P123 and nsP4, and this complex is active in negative-strand RNA synthesis (22). The latter RNA is present in the double-stranded RNA (dsRNA) replicative intermediate and is associated with plasma membrane and endosome-like vesicular organelles (8). Further processing of the polyproteins into individual nsP1 to nsP4 makes the RC capable of the synthesis of the positive-strand genome and subgenomic RNA but not of negative-strand RNA (23, 41, 42). Thus, the completely processed nsPs utilize only the promoters located on the negative strand of the viral genome.The defined promoters in the alphavirus genomes include (i) a 3′-terminal 19-nucleotide (nt)-long, conserved sequence element (CSE) adjacent to the poly(A) tail (12, 13, 19); (ii) the subgenomic promoter in the negative-strand copy of the viral genome (25); and (iii) the promoter for the synthesis of the positive-strand viral genome (45). The latter promoter is located at the 3′ end of the negative strand of the viral genome and has a complex structure. The two identified elements include the sequence, encoded by the 5′ untranslated region (5′UTR) (a core promoter) (5, 9, 32), and a 51-nt CSE, found ∼150 nt downstream of the genome''s 5′ terminus in the nsP1-encoding sequence. Our previous results and those of other research groups demonstrated that the 51-nt CSE functions as a replication enhancer in a virus- and cell-dependent mode (4, 33). Clustered mutations in the VEEV 51-nt CSE or its complete deletion either had deleterious effects on RNA replication or completely abolished RNA synthesis (30). However, RNA replication was ultimately recovered due to an accumulation of compensatory, adaptive mutations in either VEEV nsP2 or nsP3 (30). Thus, the 51-nt CSE in the VEEV genome is not absolutely essential for virus replication, but its presence is highly beneficial for achieving the most efficient growth rates in cells of both vertebrate and invertebrate origins. Alphavirus core promoters demonstrate a very low level of sequence conservation and also function in cell- and virus-specific modes (9). Previous studies suggested that the sequence and/or secondary structure of the VEEV core promoter plays a critical role in virus pathogenesis, and the G3→A (A3) mutation, found in an attenuated strain of VEEV TC-83, is one of the determinants of its less pathogenic phenotype (17, 55). However, information about functional elements of the VEEV core promoter remains incomplete, and its structural and functional elements have not yet been dissected.In this study, we applied a combination of molecular approaches to further define the functional components of the VEEV 5′UTR-specific core promoter, which mediates positive-strand genome synthesis. Our results demonstrate the presence of three structural RNA elements, two of which synergistically determine promoter activity. The first element of the promoter is a very short, 5′-terminal sequence, which is not involved in a stable secondary structure. Point mutations in the very 5′-terminal nucleotides have a deleterious effect on genome RNA replication. The second element is the short RNA stem, located in close proximity to the 5′ end of the genome. Mutations changing either the stability or sequence of the stem strongly affect virus replication and cause its rapid evolution, leading to the appearance of heterologous repeating elements in the unpaired 5′ terminus or the generation of other sequences that might potentially fold into stem structures. Surprisingly, the third structural RNA element, the loop, appears to play no important role in RNA replication and can be replaced either by a shorter loop or by the loop having a heterologous sequence without a detectable effect on virus and RNA replication.     

The Biology of Kaposi＇s Sarcoma-Associated Herpesvirus and the Infection of Human Immunodeficiency Virus

Kaposi sarcoma-associated herpesvirus (KSHV),also known as human herpesvirus 8 (HHV-8),is discovered in 1994 from Kaposi's sarcoma (KS) lesion of an acquired immunodeficiency syndrome (AIDS)patient.In addition to its association with KS,KSHV has also been implicated as the causative agent of two other AIDS-associated malignancies:primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD).KSHV is a complex DNA virus that not only has the ability to promote cellular growth and survival for tumor development,but also can provoke deregulated angiogenesis,inflammation,and modulate the patient's immune system in favor of tumor growth.As KSHV is a necessary but not sufficient etiological factor for KS,human immunodeficiency virus (HIV) is a very important cofactor.Here we review the basic information about the biology of KSHV,development of pathogenesis and interaction between KSHV and HIV.     

Assembly of the Murine Leukemia Virus Is Directed towards Sites of Cell–Cell Contact

We have investigated the underlying mechanism by which direct cell–cell contact enhances the efficiency of cell-to-cell transmission of retroviruses. Applying 4D imaging to a model retrovirus, the murine leukemia virus, we directly monitor and quantify sequential assembly, release, and transmission events for individual viral particles as they happen in living cells. We demonstrate that de novo assembly is highly polarized towards zones of cell–cell contact. Viruses assembled approximately 10-fold more frequently at zones of cell contact with no change in assembly kinetics. Gag proteins were drawn to adhesive zones formed by viral Env glycoprotein and its cognate receptor to promote virus assembly at cell–cell contact. This process was dependent on the cytoplasmic tail of viral Env. Env lacking the cytoplasmic tail while still allowing for contact formation, failed to direct virus assembly towards contact sites. Our data describe a novel role for the viral Env glycoprotein in establishing cell–cell adhesion and polarization of assembly prior to becoming a fusion protein to allow virus entry into cells.