Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

A Short N-Terminal Peptide Motif on Flavivirus Nonstructural Protein NS1 Modulates Cellular Targeting and Immune Recognition

Flavivirus NS1 is a versatile nonstructural glycoprotein, with intracellular NS1 functioning as an essential cofactor for viral replication and cell surface and secreted NS1 antagonizing complement activation. Even though NS1 has multiple functions that contribute to virulence, the genetic determinants that regulate the spatial distribution of NS1 in cells among different flaviviruses remain uncharacterized. Here, by creating a panel of West Nile virus-dengue virus (WNV-DENV) NS1 chimeras and site-specific mutants, we identified a novel, short peptide motif immediately C-terminal to the signal sequence cleavage position that regulates its transit time through the endoplasmic reticulum and differentially directs NS1 for secretion or plasma membrane expression. Exchange of two amino acids within this motif reciprocally changed the cellular targeting pattern of DENV or WNV NS1. For WNV, this substitution also modulated infectivity and antibody-induced phagocytosis of infected cells. Analysis of a mutant lacking all three conserved N-linked glycosylation sites revealed an independent requirement of N-linked glycans for secretion but not for plasma membrane expression of WNV NS1. Collectively, our experiments define the requirements for cellular targeting of NS1, with implications for the protective host responses, immune antagonism, and association with the host cell sorting machinery. These studies also suggest a link between the effects of NS1 on viral replication and the levels of secreted or cell surface NS1.West Nile virus (WNV) is a single-stranded, positive-sense enveloped RNA Flavivirus that cycles in nature between birds and Culex mosquitoes. It is endemic in parts of Africa, Europe, the Middle East, and Asia, and outbreaks occur annually in North America. More than 29,000 human cases of severe WNV infection have been diagnosed in the United States since its entry in 1999, and millions have been infected and remain undiagnosed (9). Humans can develop a febrile illness that progresses to a flaccid paralysis, meningitis, or encephalitis syndrome (59). Dengue virus (DENV) is a genetically related flavivirus that is transmitted by Aedes aegypti and Aedes albopictus mosquitoes and causes clinical syndromes in humans, ranging from an acute self-limited febrile illness (dengue fever [DF]) to a severe and life-threatening vascular leakage and bleeding diathesis (dengue hemorrhagic fever/dengue shock syndrome [DHF/DSS]). Globally, DENV causes an estimated 50 million infections annually, resulting in 500,000 hospitalizations and ∼22,000 deaths (45).The ∼10.7-kb Flavivirus RNA genome is translated as a single polyprotein, which is then cleaved into three structural proteins (C, prM/M, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by virus- and host-encoded proteases (39). The multifunctional NS proteins include an RNA-dependent RNA polymerase and methyltransferase (NS5), a helicase and protease (NS3), accessory proteins that form part of the viral replication complex, and immune evasion molecules (33, 34). Flavivirus NS1 is a 48-kDa nonstructural glycoprotein with two or three N-linked glycans, depending on the flavivirus, and is absent from the virion. The Japanese encephalitis virus (JEV) serogroup (West Nile, Japanese, Murray Valley, and St. Louis encephalitis viruses) generate NS1 and NS1′ proteins, the latter of which is a product of a ribosomal frameshift event that occurs at a heptanucleotide motif located at the beginning of the NS2A gene (25, 47).NS1 is an essential gene as it is required for efficient viral RNA replication (34, 41, 44). In infected mammalian cells, NS1 is synthesized as a soluble monomer, dimerizes after posttranslational modification in the lumen of the endoplasmic reticulum (ER), and accumulates extracellularly as higher-order oligomers, including hexamers (16, 26, 64, 65). Soluble NS1 binds back to the plasma membrane of uninfected cells through interactions with sulfated glycosaminoglycans (5). In infected cells, NS1 is also directly transported to and expressed on the plasma membrane although it lacks a transmembrane domain or canonical targeting motif. The mechanism of cell surface expression of flavivirus NS1 in infected cells remains uncertain although some fraction may be linked through an atypical glycosyl-phosphatidylinositol anchor (30, 50) or lipid rafts (49).NS1 has been implicated in having pathogenic consequences in flavivirus infection. The high levels of NS1 in the serum of DENV-infected patients correlate with severe disease (4, 37). NS1 has been proposed to facilitate immune complex formation (4), elicit auto-antibodies that react with host matrix proteins (21), damage endothelial cells via antibody-dependent complement-mediated cytolysis (38), or directly enhance infection (1). Flavivirus NS1 also has direct immune evasion functions and antagonizes complement activation on cell surfaces and in solution. WNV NS1 attenuates the alternative pathway of complement activation by binding the complement-regulatory protein factor H (11, 36), and DENV, WNV, and YFV NS1 proteins bind C1s and C4 in a complex to promote efficient degradation of C4 to C4b (3).Although NS1 is absent from the virion, antibodies against it can protect against infection in vivo. Immunization with purified NS1 or passive administration of some anti-WNV, anti-yellow fever virus (YFV), and anti-DENV NS1 monoclonal antibodies (MAbs) protect mice against lethal virus challenge (12, 13, 17, 22, 27, 29, 31, 32, 56-58). Initial studies with isotype switch variants and F(ab′)₂ fragments of anti-YFV NS1 MAbs suggested that the Fc region of anti-NS1 MAbs was required for protection (58). Subsequent mechanistic studies with WNV NS1 indicated that only MAbs recognizing cell surface-associated NS1 trigger Fc-γ receptor I- and/or IV-mediated phagocytosis and clearance of infected cells (13).In this study, we identify a reciprocal relationship between the secretion and cell surface expression patterns of WNV and DENV NS1s. Using WNV-DENV NS1 chimeras and point mutants, we identified a novel short peptide motif immediately C-terminal to the signal sequence cleavage position that directs NS1 for secretion or to the plasma membrane. These studies begin to explain how NS1 regulates its localization to several cellular compartments (ER, cell surface, and extracellular space) and have implications for viral infectivity, association with the host cell sorting machinery, and protective immune responses.     

Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.Since the first detection of the Asian lineage of highly pathogenic avian influenza (HPAI) virus (H5N1) in southern China in 1996, H5N1 virus infection in birds has continued for 13 years in Asia, acquiring pathogenicity not only in birds but also in mammals. In 1997, the H5N1 Hong Kong isolates caused illness and death in a variety of terrestrial birds and even in humans (9, 37, 48, 49). In 2001, emerging H5N1 Hong Kong isolates were more pathogenic to chickens and the mean death time (MDT) was 2 days without any prior clinical signs (12). In 2003 to 2004, the H5N1 epizootic occurred simultaneously in East Asian countries (22, 30). The 2003/2004 H5N1 isolates caused death in taxonomically diverse avian species, including domestic ducks (46, 47, 51), and humans (7, 55). Furthermore, the first indication of wild aquatic bird involvement occurred at recreational parks in Hong Kong in late 2002 to 2003 (46), and then migratory aquatic bird die-off occurred in 2005 at Qinghai Lake in China (6, 24). The broad host spectrum and increased pathogenicity of H5N1 viruses to diverse bird species raise serious concerns about the worldwide spread of the virus by migratory birds.According to the international criteria, HPAI viruses are defined by over 75% mortality in 4- to 8-week-old chickens following an intravenous pathogenicity test or an intravenous pathogenicity index (IVPI) of more than 1.2 in 6-week-old chickens (34); however, there are some variations in pathogenicity intensity among the HPAI viruses in chickens (1, 3, 5, 12, 15, 28, 31, 48, 50-52, 57). Most of the HPAI viruses that were isolated before 1996 cause severe clinical signs (e.g., ruffled feathers, depression, labored breathing, and neurological signs) and severe gross lesions (e.g., head and face edema, cyanosis, subcutaneous hemorrhages in combs and leg shanks, and necrosis of combs and wattles) in chickens (1, 3, 15, 31, 50, 52, 57). These viruses usually kill chickens 3 to 6 days after intranasal inoculation. On the other hand, the recently emerged Asian H5N1 HPAI viruses are more virulent and kill chickens within 1 to 2 days without causing typical clinical signs and gross lesions (5, 12, 27, 33, 48, 51), although some Asian H5N1 viruses, such as A/Goose/Guangdong/2/96 (23), A/goose/Hong Kong/437-10/99 (17), and A/chicken/Indonesia/7/03 (58), were less virulent. To successfully control HPAI in poultry, it is important to better understand the mechanisms of increased pathogenicity of recent H5N1 HPAI viruses in chickens.The Asian H5N1 HPAI virus has another important characteristic, which is its capability of crossing host-species barriers. It was reported that the H5N1 virus can infect and cause death in mammals such as mice (5, 9, 12, 14, 29), cats (21), tigers (2), ferrets (11, 26), monkeys (40), and humans (7, 49, 55). High-level inductions of proinflammatory cytokines in mammals infected with the H5N1 viruses, referred to as “cytokine storms,” have been hypothesized to contribute to the severity of pathological changes and ultimate death (4, 7, 13, 45, 55). Cytokine and chemokine dysregulation was detected in clinical cases of H5N1-infected humans (8, 13, 36) and also in monkeys experimentally infected with the H1N1 Spanish flu strain (20). In a mouse model, lymphocyte apoptosis and cytokine dysregulation have been proposed to contribute to the severity of the disease caused by H5N1 (56). Investigations with transgenic mice deficient in each cytokine gene suggest that tumor necrosis factor alpha (TNF-α) may contribute to morbidity and interleukin-1 (IL-1) may be important for virus clearance (53). However, mice deficient in TNF-α or IL-6 succumb to infection with H5N1, and cytokine inhibition treatment does not prevent death (42), suggesting that therapies targeting the virus rather than cytokines may be preferable. Thus, the significance of elevated proinflammatory cytokine responses in the pathogenesis of H5N1-infected mammals requires further studies.In contrast, little is known about proinflammatory cytokine responses and their roles in pathogenicity in chickens infected with HPAI viruses, including the recent Asian H5N1 viruses. It was reported that infection with an HPAI virus results in upregulation of gene expression of gamma interferon (IFN-γ) and inducible nitric oxide synthase (58). However, the roles of proinflammatory cytokines in disease severity and outcomes in chickens infected systemically with HPAI viruses are largely unknown. The less-virulent Asian H5N1 virus, which causes severe clinical signs and gross lesions in chickens (17, 23, 27, 58), would be a valuable tool for investigating the role of proinflammatory cytokines in chickens infected with HPAI viruses, as well as for exploring the pathogenesis of the more-virulent Asian H5N1 HPAI virus, because of the antigenic and molecular similarities between them.In this study, we compared the pathogenicities in chickens of the less-virulent and more-virulent Asian H5N1 HPAI viruses based on MDT, fever, cytokine responses, and viral replication. Our results suggest that the shift in the Asian H5N1 virus to increased virulence may be associated with efficient and rapid replication of the virus in chickens, accompanied by early destruction of host immune responses and followed by peracute death before fever can be induced. Finally, we discuss candidate genes that may account for the high pathogenicity of Asian H5N1 HPAI viruses in chickens.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).     

A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity

For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.The pestiviruses Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, Classical swine fever virus (CSFV), and Border disease virus (BDV) are causative agents of economically important livestock diseases. Together with the genera Flavivirus, including several important human pathogens like Dengue fever virus, West Nile virus, Yellow fever virus, and Tick-borne encephalitis virus, and Hepacivirus (human Hepatitis C virus [HCV]), the genus Pestivirus constitutes the family Flaviviridae (8, 20). All members of this family are enveloped viruses with a single-stranded positive-sense RNA genome encompassing one large open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions (NTR) (see references 8 and 28 for reviews). The ORF encodes a polyprotein which is co- and posttranslationally processed into the mature viral proteins by viral and cellular proteases. For BVDV, the RNA genome is about 12.3 kb in length and encodes a polyprotein of about 3,900 amino acids. The first third of the ORF encodes a nonstructural (NS) autoprotease and four structural proteins, while the remaining part of the genome encodes NS proteins which share many common characteristics and functions with the corresponding NS proteins encoded by the HCV genome (8, 28). NS2 of BVDV represents a cysteine autoprotease which is distantly related to the HCV NS2-3 protease (26). NS3, NS4A, NS4B, NS5A, and NS5B are essential components of the pestivirus replicase (7, 10, 49). NS3 possesses multiple enzymatic activities, namely serine protease (48, 52, 53), NTPase (46), and helicase activity (51). NS4A acts as an essential cofactor for the NS3 proteinase. NS5B represents the RNA-dependent RNA polymerase (RdRp) (22, 56). The functions of NS4B and NS5A remain to be determined. NS5A has been shown to be a phosphorylated protein that is associated with cellular serine/threonine kinases (44).According to their effects in tissue culture, two biotypes of pestiviruses are distinguished: cytopathogenic (cp) and noncytopathogenic (ncp) viruses (17, 27). The occurrence of cp BVDV in cattle persistently infected with ncp BVDV is directly linked to the induction of lethal mucosal disease in cattle (12, 13). Previous studies have shown that cp BVDV strains evolved from ncp BVDV strains by different kinds of mutations. These include RNA recombination with various cellular mRNAs, resulting in insertions of cellular protein-coding sequences into the viral genome, as well as insertions, duplications, and deletions of viral sequences, and point mutations (1, 2, 9, 24, 33, 36, 37, 42). A common consequence of all these genetic changes in cp BVDV genomes is the efficient production of NS3 at early and late phases of infection. In contrast, NS3 cannot be detected in cells at late time points after infection with ncp BVDV. An additional major difference is that the cp viruses produce amounts of viral RNA significantly larger than those of their ncp counterparts (7, 32, 50). While there is clear evidence that cell death induced by cp BVDV is mediated by apoptosis, the molecular mechanisms involved in pestiviral cytopathogenicity are poorly understood. In particular, the role of NS3 in triggering apoptosis remains unclear. It has been hypothesized that the NS3 serine proteinase might be involved in activation of the apoptotic proteolytic cascade (21, 55). Furthermore, it has been suggested that the NS3-mediated, enhanced viral RNA synthesis of cp BVDV and subsequently larger amounts of viral double-stranded RNAs may play a crucial role in triggering apoptosis (31, 54).In this study, we describe generation and characterization of a temperature-sensitive (ts) cp BVDV mutant whose ability to cause viral cytopathogenicity at high temperature is strongly attenuated. Our results demonstrate that a single amino acid substitution in NS2 attenuates BVDV cytopathogenicity at high temperature without affecting production of infectious viruses and expression of NS3 in a temperature-dependent manner.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Virulence Determinants of Avian H5N1 Influenza A Virus in Mammalian and Avian Hosts: Role of the C-Terminal ESEV Motif in the Viral NS1 Protein

We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain- and host-dependent manner.The transmission of highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype to humans since the year 1997 has caused a high mortality rate of almost 60% (62). Patients infected with H5N1 influenza virus developed mainly severe respiratory disease, characterized by fever, cough, shortness of breath, and pneumonia, that frequently progressed to acute respiratory distress syndrome (ARDS) and multiorgan failure (28, 68, 69). In fatal cases, the median time from onset to death was 9 to 10 days (1). Systemic spread (18) and hypercytokinemia (11) have been described as possible disease-aggravating factors of HPAIV-H5N1 viruses, but the reasons for their high virulence in humans are incompletely understood.Due to the potential pandemic threat presented by H5N1 viruses, there is great interest in the identification of viral virulence determinants and their mode of action. This is critical not only for a better understanding of the pathogenic mechanisms induced by these viruses but also for the development of new drugs to treat the infections. The high virulence of HPAIV-H5N1 isolates in the avian host correlates with the presence of a polybasic cleavage site in the hemagglutinin (HA), facilitating its intracellular cleavage by furin-like proteases (27, 50). Further, amino acid substitutions in the PA protein (T515A) (30) and in the NS1 protein (V149A) (40) have been reported to regulate the virulence of corresponding HPAIV-H5N1 isolates in ducks and chickens. The known molecular determinants of virulence in mammalian hosts also include the polybasic cleavage site in the HA (23) and several polymorphisms in the PB2 polymerase subunit and the proapoptotic PB1-F2 protein. Thus, a serine residue at position 66 in the PB1-F2 protein increased viral replication and decreased survival in the mouse model (9). Also, specific amino acid polymorphisms within PB2 (E627K or D701N) can increase virulence in mice (23, 39) and viral replication in mammalian cells (7, 57, 58). Furthermore, the nonstructural NS1 protein, which has a major function in the inhibition of type I interferon (IFN) (17, 19) and in the limitation of the antiviral effects of IFN-induced proteins, including PKR (4, 22), OAS/RNase L (45), and RIG-I (16, 48, 63, 64), contributes to virulence in mammals (34, 55).The domain structure of the NS1 protein is well characterized; it includes an N-terminal RNA binding and dimerization domain and a nuclear localization signal (NLS) at positions 34 to 38 (summarized in reference 19). The NS1 proteins of most human strains circulating between 1950 and 1986 also contain a second NLS at positions 219 to 227 (NLS-2), which includes four conserved basic amino acids (K219, R220, R224, R227) (44). A large-scale sequence analysis showed that the NS1 proteins of avian and human influenza viruses differ in their C-terminal sequences, indicating possible differences in the associated activity (46). Among most high- and low-pathogenicity avian influenza viruses, the last four NS1 amino acids consist of the conserved sequence ESEV (3,007 of 3,692 isolates described in the NCBI database [3]), while for the majority of seasonal human influenza viruses, the motif RSKV is typical (1,911 of 2,713 isolates). Significantly, only the NS1 protein carrying the “avian” ESEV motif interacted in vitro with 24 cellular factors carrying a PDZ (postsynaptic density protein 95, Drosophila disc large tumor suppressor, and zonula occludens 1 protein) domain. The human genome encodes at least 214 proteins containing one or more of these protein interaction modules that recognize short peptide motifs, which are most often present at the C termini of their targets (36, 38). Many PDZ domain proteins have been shown to mediate the formation and localization of higher-order complexes and to participate in various cellular signaling events regulating, for instance, cell polarity and neuronal function (31). Therefore, it was hypothesized that the abundant expression of “avian” NS1 protein capable of interacting with human PDZ domains could possibly disturb their function and aggravate disease severity in H5N1 infections (46). However, there is only limited experimental support for the universal validity of this hypothesis. The grafting of the “avian” ESEV sequence into the C terminus of NS1 protein expressed by mouse-adapted influenza A/WSN/33 virus (H1N1) decreased the mean lethal dose by about 1 order of magnitude (32). Still, it is not clear to what extent this motif contributes to the virulence of HPAIV-H5N1 and other natural influenza A viruses in avian and mammalian hosts.The goal of the present study was to elucidate the role of the C-terminal NS1 motif in viral replication and disease caused by the prototypic influenza A/Vietnam/1203/04 (VN/1203) virus, isolated in a fatal human case (60). This virus expresses an NS1 protein that is very similar or identical at positions 1 to 215 to homologues expressed by other HPAIV-H5N1 strains but naturally lacks the 10 C-terminal amino acids (aa), including the terminal ESEV motif, due to a premature stop codon (Fig. ​(Fig.1).1). We used reverse genetics to produce a recombinant VN/1203 wild-type (WT) virus and two variants with reconstituted NS1 C termini ending either with the “avian” ESEV or with the “human” RSKV sequence. Experimental infections of mice and chickens revealed that all three viruses caused highly lethal infections in both species, with only moderate differences in viral titers in the organs of the mice. Thus, we show that the C-terminal ESEV motif of the NS1 protein contributes little to the virulence of H5N1 viruses in mice and chickens, and we suggest that this motif modulates viral virulence in a strain- and host-dependent manner.Open in a separate windowFIG. 1.Growth kinetics of recombinant VN/1203 viruses expressing WT or elongated NS1 proteins in human, murine, and avian cells. (A) Scheme of the viral VN/1203-NS1 protein with the RNA binding domain and the nuclear localization signals (NLS) at positions 34 to 38 and 214 to 225 indicated. Amino acids involved in NLS2 function are underlined. The C-terminal sequences of the WT and elongated mutant NS1 proteins are given, and the PL motif is shown in boldface. (B to E) Human A549 alveolar cells, murine NIH 3T3 fibroblasts, chicken embryo fibroblasts (CEFs), or EFB-R1 duck embryo fibroblasts (DEFs) were infected with recombinant VN/1203-WT, -ESEV, or -RSKV viruses at an MOI of 0.001. Aliquots of supernatants were harvested at the indicated time points, and samples were titrated by plaque assays in MDCK cells. (F) Human A549 cells were infected at an MOI of 2, and virus titers in supernatants taken at the indicated time points were determined by plaque assays. Results are averages for at least two independent experiments with biological duplicates. Error bars indicate standard deviations.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Breadth of Neutralizing Antibodies Elicited by Stable,Homogeneous Clade A and Clade C HIV-1 gp140 Envelope Trimers in Guinea Pigs

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.The development and evaluation of novel HIV-1 Env immunogens are critical priorities of the HIV-1 vaccine field (2, 10, 25). The major antigenic target for neutralizing antibodies (NAbs) is the trimeric Env glycoprotein on the virion surface (4, 18, 30). Monomeric gp120 immunogens have not elicited broadly reactive NAbs in animal models (5, 13, 28, 29) or humans (16, 31), and thus several groups have focused on generating trimer immunogens that better mimic the native Env spike found on virions (3, 7, 14, 15, 20, 22, 27). It has, however, proven difficult to produce stable and conformationally homogeneous Env trimers. Strategies to modify Env immunogens have therefore been explored, including the removal of the cleavage site between gp120 and gp41 (3, 7, 23, 39, 40), the incorporation of an intramolecular disulfide bond to stabilize cleaved gp120 and gp41 moieties (6), and the addition of trimerization motifs such as the T4 bacteriophage fibritin “fold-on” (Fd) domain (8, 17, 39).Preclinical evaluation of candidate Env immunogens is critical for concept testing and for the prioritization of vaccine candidates. Luciferase-based virus neutralization assays with TZM.bl cells (21, 24) have been developed as high-throughput assays that can be standardized (26). However, the optimal use of this assay requires the generation of standardized virus panels derived from multiple clades that reflect both easy-to-neutralize (tier 1) and primary isolate (tier 2) viruses (21, 24). A tiered approach for the evaluation of novel Env immunogens has been proposed, in which tier 1 viruses represent homologous vaccine strains and a small number of heterologous neutralization-sensitive viruses while tier 2 viruses provide a greater measure of neutralization breadth for the purpose of comparing immunogens (24).We screened a large panel of primary HIV-1 isolates for Env stability and identified two viruses, CZA97.012 (clade C) (32) and 92UG037.8 (clade A) (17), that yielded biochemically homogeneous and stable Env trimers with well defined and uniform antigenic properties (17). The addition of the T4 bacteriophage fibritin “fold-on” (Fd) trimerization domain further increased their yield and purity (17). In the present study, we assessed the immunogenicity of these stable clade A and clade C gp140 trimers in guinea pigs. Both trimers elicited high-titer binding antibody responses and cross-clade neutralization of select tier 1 viruses as well as low-titer but detectable NAb responses against select tier 2 viruses from clades A, B, and C. These data demonstrate the immunogenicity of these stable gp140 trimers and highlight the utility of standardized virus panels in the evaluation of novel HIV-1 Env immunogens.     

Identification of a Feline Leukemia Virus Variant That Can Use THTR1, FLVCR1, and FLVCR2 for Infection

The pathogenic subgroup C feline leukemia virus (FeLV-C) arises in infected cats as a result of mutations in the envelope (Env) of the subgroup A FeLV (FeLV-A). To better understand emergence of FeLV-C and potential FeLV intermediates that may arise, we characterized FeLV Env sequences from the primary FY981 FeLV isolate previously derived from an anemic cat. Here, we report the characterization of the novel FY981 FeLV Env that is highly related to FeLV-A Env but whose variable region A (VRA) receptor recognition sequence partially resembles the VRA sequence from the prototypical FeLV-C/Sarma Env. Pseudotype viruses bearing FY981 Env were capable of infecting feline, human, and guinea pig cells, suggestive of a subgroup C phenotype, but also infected porcine ST-IOWA cells that are normally resistant to FeLV-C and to FeLV-A. Analysis of the host receptor used by FY981 suggests that FY981 can use both the FeLV-C receptor FLVCR1 and the feline FeLV-A receptor THTR1 for infection. However, our results suggest that FY981 infection of ST-IOWA cells is not mediated by the porcine homologue of FLVCR1 and THTR1 but by an alternative receptor, which we have now identified as the FLVCR1-related protein FLVCR2. Together, our results suggest that FY981 FeLV uses FLVCR1, FLVCR2, and THTR1 as receptors. Our findings suggest the possibility that pathogenic FeLV-C arises in FeLV-infected cats through intermediates that are multitropic in their receptor use.Feline leukemia viruses (FeLVs) are pathogenic retroviruses of domestic cats that induce proliferative, degenerative, and immunosuppressive disorders (17, 18, 27). Infected cats often contain a mixture of FeLVs that have been categorized into subgroups A, B, and C based on their interference and in vitro host range properties (39). These subgroups have been shown to be tightly associated with distinct feline diseases (17, 27). A fourth subgroup (T) that is highly related to FeLV-A has also been reported (26) and has been shown to be associated with immune deficiency (30). FeLV-A is the primary strain that is transmitted between cats and is the progenitor virus from which other subgroups arise. FeLV-B arises by recombination between endogenous retroviral sequences present in the cat genome and the gene encoding the FeLV-A envelope (Env) protein (32, 40, 42) that is responsible for host cell surface receptor recognition. FeLV-C and FeLV-T are formed by mutations in the FeLV-A Env gene (10, 27, 38). Env recombination and mutations are a major determinant of the host receptors used by the different FeLV subgroups (3, 23, 34, 46, 47).The emergence of pathogenic FeLV-C in cats infected with FeLV-A provides a classic example of Env mutations that switch the host receptor used for infection, leading to a fatal pathogenic disease in the host. The emergence of FeLV-C is tightly associated with red blood cell aplasia, a fatal feline anemia characterized by a specific disruption in erythroid progenitor cell development (2, 12, 19, 28). Moreover, FeLV-C emergence coincides with a switch in the host receptor used for infection from the thiamine transporter THTR1 (FeLV-A receptor) (23) to the heme exporter FLVCR1 (FeLV-C receptor) (34, 35, 46). Previous studies have suggested that the feline anemia is caused by the FeLV-C Env protein binding to, and disrupting, the cellular function of FLVCR1 (1, 34, 46). Indeed, expression of FeLV-C Env in hematopoietic stem cells (36) or disruption of FLVCR1 (21, 35) specifically disrupts early erythropoiesis, which mimics the anemia observed in cats with FeLV-C. Interestingly, the emergence of FeLV-C from FeLV-A is analogous to the emergence of cytopathic X4 strains of human immunodeficiency virus type 1 (HIV-1) in individuals infected with the R5 strain of HIV-1. Analogous to the emergence of FeLV-C, X4 HIV-1 arises by Env mutations in the R5 HIV-1 strain, which leads to a switch in the coreceptor used for infection allowing an expansion in HIV-1 cell tropism and subsequently to accelerated AIDS (4, 8). However, whereas HIV-1 pathogenesis also involves emergence of variants or X4/R5 intermediates that can use multiple related coreceptors for infection (5, 8, 11), the mechanism of how FeLV-C emerges, in terms of the presence of FeLV-A/FeLV-C intermediates, has yet to be elucidated.To better understand the emergence of pathogenic FeLV-C in infected cats and potential FeLV variants/intermediates that may arise, we isolated and characterized FeLV Env sequences from a primary FeLV isolate derived from a cat with pure red cell aplasia. In this study, we report the isolation and characterization of the novel FY981 Env, a hybrid FeLV-A/FeLV-C Env that, when pseudotyped, can use FLVCR1, THTR1, and the FLVCR1-related FLVCR2 for infection. Our findings suggest that the FY981 Env could represent a potential FeLV intermediate that arises during the emergence of pathogenic FeLV-C.