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.     

Comparison of the 3′ Termini of Discrete Segments of the Double-Stranded Ribonucleic Acid Genomes of Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

The 3' terminal nucleosides of the isolated components of double-stranded ribonucleic acids of reovirus, wound tumor virus, and cytoplasmic polyhedrosis virus were determined by labeling with tritiated sodium borohydride. All wound tumor virus and cytoplasmic polyhedrosis virus components appear to contain approximately equal amounts of U(OH) and C(OH) termini. Reovirus segments have essentially only C(OH) termini.     

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.     

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.     

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.     

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.     

Refinement and Analysis of the Mature Zika Virus Cryo-EM Structure at 3.1 Å Resolution

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Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.     

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.     

Impairment of the Antibody-Dependent Phagocytic Function of PMNs through Regulation of the FcγRs Expression after Porcine Reproductive and Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome (PRRS) is identified as one of the most important etiological agents in multifactorial respiratory disease of swine and can predispose pigs to secondary infections by other pathogens, usually bacteria. To understand the mechanism for an increased susceptibility to secondary bacterial infections, we investigated the antibody-dependent phagocytosis behaviour and killing ability of PMNs after infection by PRRSV strains BJ-4 or HN07-1. PMN’s antibody-dependent phagocytosis and their ability to kill E.coli were both noticeably decreased following PRRSV infection, in particular with the highly pathogenic strain HN07-1. As the change in this function of the PMNs may reflect a variation in the expression of FcγRs, the expression profiles of the activating and the inhibitory FcγRs were examined. We found that RNA expression of the inhibitory receptor FcγRIIB was up-regulated post-infection, and this was greater after infection with the more virulent PRRSV strain HN07-1. The activating receptor FcγRIIIA RNA expression was on the other hand inhibited to the same extent by both PRRSV strains. Neutralizing antibody titers post-infection by PRRSV strains BJ-4 or HN07-1 were also detected. All of the pigs in infection groups showed viraemia by the end of the study (56 DPI). These observations may help to understand the mechanism of increased susceptibility to secondary bacterial infections following PRRSV infection.