Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

Ehrlichia ruminantium infection (heartwater) in wild animals

Several wild animal species have been implicated as hosts of Ehrlichia ruminantium (formerly Cowdria ruminantium), the rickettsial agent causing heartwater, a fatal disease of domestic ruminants in sub-Saharan Africa and eastern Caribbean. However, evidence for infection in most wild species is inconclusive because of inadequate diagnostic techniques. Infection has been proven only in 12 African ruminants, three non-African ruminants and two African rodents. A subclinical carrier state occurs in eight of the African ruminant species. Further studies on E. ruminantium infection in wild animal species are needed in order to determine the host range of this pathogen accurately. The host range of Ehrlichia ruminantium in wildlife is reviewed here and the role played by these species in the epidemiology and spread of heartwater is discussed.     

Prevalence of neutralizing antibodies to bovid herpesvirus 2 in African wildlife

A total of 3,470 sera, collected between 1963 and 1980 from 45 different species of wildlife in nine African countries, was examined for virus neutralizing (VN) antibodies to bovid herpesvirus 2. Antibodies were demonstrated in 20 species including 15 Bovidae, two Suidae, hippopotamus (Hippopotamus amphibius), giraffe (Giraffa camelopardalis) and a green monkey (Cercopithecus aethiops); 11 of these species had not been previously recorded as sero-positive. Although the significance of neutralizing antibodies in the absence of virus isolation remains in doubt, results suggest that infection is widespread in wildlife. The highest VN titres were recorded in waterbuck (Kobus ellipsiprymnus and K. defassa), reedbuck (Redunca arundinum) and buffalo (Syncerus caffer). Infection appears to be continuous in free-living populations of buffalo and antibodies are present in the majority of animals by the age of 2 yr.     

Zoonotic Potential of Simian Arteriviruses

Wild nonhuman primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae. Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild nonhuman primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be “preemergent” zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur.     

Virus infections in wild plant populations are both frequent and often unapparent

? Premise of the study: Pathogens are thought to regulate host populations. In agricultural crops, virus infection reduces yield. However, in wild plants little is known about the spatial and temporal patterns of virus prevalence. Thus, pathogen effects on plant population dynamics are unclear. Prevalence data provide necessary background for (1) evaluating the effects of virus infection on plant population size and dynamics and (2) improving risk assessment of virus-resistant transgenic crops. ? Methods: We used ELISA and RT-PCR to survey wild Cucurbita pepo populations over 4 years for five viruses, aphid-transmitted viruses of the genus Potyvirus as a group and PCR to survey for virus-resistance transgenes. In addition, we surveyed the literature for reports of virus prevalence in wild populations. ? Key results: In 21 C. pepo populations, virus prevalence (0-74%) varied greatly among populations, years, and virus species. In samples analyzed by both ELISA and RT-PCR, RT-PCR detected 6-44% more viruses than did ELISA. Eighty percent of these infections did not cause any visually apparent symptoms. In our samples, the virus-resistance transgene was not present. In 30 published studies, 92 of 146 tested species were infected with virus, and infection rates ranged from 0.01-100%. Most published studies used ELISA, suggesting virus prevalence is higher than reported. ? Conclusions: In wild C. pepo, the demographic effects of virus are likely highly variable in space and time. Further, our literature survey suggests that such variation is probably common across plant species. Our results indicate that risk assessments for virus-resistant transgenic crops should not rely on visual symptoms or ELISA and should include data from multiple populations over multiple years.     

Growth Kinetics of Yaba Tumor Poxvirus After In Vitro Adaptation to Cercopithecus Kidney Cells

Yaba tumor poxvirus has been adapted to continuous in vitro cultivation in monolayers of cercopithecus kidney cells. At 35 C, the minimum replicative cycle, after synchronous infection of CV-1 cells with multiplicity of infection of 135 focusforming units per cell, was 35 hr; however, maximum virus yields were not obtained until 75 hr postinfection (PI). Cytoplasmic incorporation of (3)H-thymidine [viral deoxyribonucleic acid (DNA) synthesis] was detected 3 hr PI and was preceded by synthesis of nonstructural associated antigens (YS). Synthesis of YS antigens was not inhibited by the DNA inhibitor, arabinofuranosyl cytosine (ARA-C). Synthesis of at least two virion structural antigens, although not detected by immunofluorescence until 2 hr after the onset of DNA synthesis, occurred in the presence of ARA-C, indicating potential translation of these structural antigens from parental DNA. The first progeny DNA was completed by 20 hr PI but was not detected in infectious form until 35 hr PI. The maximum rate of progeny DNA completion occurred between 20 and 30 hr PI. DNA synthesis continued 45 to 50 hr PI. The adapted virus retained its oncogenicity and, like the wild type, replicated better at 35 C than at 37 C. A synthetic step associated with viral DNA synthesis appears to be temperature-sensitive.     

Rabies Virus and Canine Distemper Virus in Wild and Domestic Carnivores in Northern Kenya: Are Domestic Dogs the Reservoir?

Rabies virus (RV) and canine distemper virus (CDV) can cause significant mortality in wild carnivore populations, and RV threatens human lives. We investigated serological patterns of exposure to CDV and RV in domestic dogs (Canis familiaris), African wild dogs (Lycaon pictus), black-backed jackals (Canis mesomelas), spotted hyenas (Crocuta crocuta), striped hyenas (Hyaena hyaena) and African lions (Panthera leo), over a 10-year period, in a Kenyan rangeland to assess the role domestic dogs may play in the transmission dynamics of these two important canid pathogens. Observed patterns of RV exposure suggested that repeated introduction, rather than maintenance, occurred in the wild carnivore species studied. However, RV appeared to have been maintained in domestic dogs: exposure was more likely in domestic dogs than in the wild carnivores; was detected consistently over time without variation among years; and was detected in juveniles (≤1-year-old) as well as adults (>1-year-old). We conclude that this domestic dog population could be a RV reservoir. By contrast, the absence of evidence of CDV exposure for each carnivore species examined in the study area, for specific years, suggested repeated introduction, rather than maintenance, and that CDV may require a larger reservoir population than RV. This reservoir could be a larger domestic dog population; another wildlife species; or a “metareservoir” consisting of multiple interconnected carnivore populations. Our findings suggest that RV risks to people and wild carnivores might be controlled by domestic dog vaccination, but that CDV control, if required, would need to target the species of concern.     

Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection

Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an anti-apoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.     

Experimental avian PMV-2 infection in a domesticated wild host: daily behavior and effect on activity levels

Paramyxovirus type 2 (PMV-2) isolated from wild birds is often considered non-pathogenic, but nothing is known about its effects on overall behavior and fitness of free-flying birds. Domestically bred, African cut-throat finches (Amadina fasciata), a species from which PMV-2 has been isolated in the wild, were inoculated with a Central American field strain of PMV-2. Patterns of behavior were examined before and after viral challenge to quantify inapparent, sublethal effects of the disease. Infected birds demonstrated a significant decrease in activity (P = 0.01) followed by an apparent recovery period. Antibody titers confirmed infection in inoculated birds and indicated that sentinel birds did not become infected.     

The host restriction factor APOBEC3G and retroviral Vif protein coevolve due to ongoing genetic conflict

APOBEC3G (A3G) is a host cytidine deaminase that inhibits retroviruses. HIV and related primate lentiviruses encode Vif, which counteracts A3G by inducing its degradation. This Vif-mediated A3G inhibition is species specific, suggesting that the A3G-Vif interaction has evolved as primate lentiviruses have adapted to their hosts. We examined the evolutionary dynamics of the A3G-Vif interaction within four African green monkey (AGM) subspecies, which are each naturally infected with a distinct simian immunodeficiency virus (SIV). We identified single amino acid changes within A3G in two AGM subspecies that render it resistant to Vif proteins, except for Vif from the viruses that naturally infect these subspecies. Moreover, experimental infection of AGMs shows that Vif can rapidly adapt to these arising Vif-resistant A3G genotypes. These data suggest that despite being generally nonpathogenic in its natural host, SIV infection selects for Vif-resistant forms of A3G in AGM populations, driving Vif counterevolution and functional divergence.     

Low susceptibility of Achatina fulica from Brazil to infection with AngioIylus costaricensis and A. cantonensis

Introduction of Achatina fulica in Brazil has led to serious concerns about its role as vector for metaIylid worms: AngioIylus costaricensis and A. cantonensis. Experimental infection with both parasites was performed to evaluate the potential risk for their transmission by the giant African snail. Groups of 5 animals, both wild and bred at captivity were exposed at different inocula: 1, 5, and 10 x 10(3) L1 of A. costaricensis and A. cantonensis. In all groups, few snails got infected and parasitic burden was low. Two different ways of infection were tested: ingestion produced higher numbers of L3 than the inoculation through an artificial hole in the shell. We also report the parasitological examination of 6 batches of wild A. fulica from Florianópolis, state of Santa Catarina, Brazil: only 1 out of 244 animals were infected with metaIylid larvae. Taken together these data indicate that the giant African snail occurring in Southern Brazil is not a permissive host for both AngioIylus species and does not represent a significant risk for transmission of these parasites.     

Phosphatidylinositol-3-kinase (PI3K) is activated by influenza virus vRNA via the pathogen pattern receptor Rig-I to promote efficient type I interferon production

The phosphatidylinositol-3-kinase (PI3K) was identified to be activated upon influenza A virus (IAV) infection. An early and transient induction of PI3K signalling is caused by viral attachment to cells and promotes virus entry. In later phases of infection the kinase is activated by the viral NS1 protein to prevent premature apoptosis. Besides these virus supporting functions, it was suggested that PI3K signalling is involved in dsRNA and IAV induced antiviral responses by enhancing the activity of interferon regulatory factor-3 (IRF-3). However, molecular mechanisms of activation remained obscure. Here we show that accumulation of vRNA in cells infected with influenza A or B viruses results in PI3K activation. Furthermore, expression of the RNA receptors Rig-I and MDA5 was increased upon stimulation with virion extracted vRNA or IAV infection. Using siRNA approaches, Rig-I was identified as pathogen receptor necessary for influenza virus vRNA sensing and subsequent PI3K activation in a TRIM25 and MAVS signalling dependent manner. Rig-I induced PI3K signalling was further shown to be essential for complete IRF-3 activation and consequently induction of the type I interferon response. These data identify PI3K as factor that is activated as part of the Rig-I mediated anti-pathogen response to enhance expression of type I interferons.     

Influenza A virus NS1 protein activates the PI3K/Akt pathway to mediate antiapoptotic signaling responses

Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3beta and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.     

Susceptibility of wood ducks to H5N1 highly pathogenic avian influenza virus

Since 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses have caused mortality in numerous species of wild birds; this is atypical for avian influenza virus (AIV) infections in these avian species, especially for species within the order Anseriformes. Although these infections document the susceptibility of wild birds to H5N1 HPAI viruses and the spillover of these viruses from infected domestic birds to wild birds, it is unknown whether H5N1 HPAI viruses can persist in free-living avian populations. In a previous study, we established that wood ducks (Aix sponsa) are highly susceptible to infection with H5N1 HPAI viruses. To quantify this susceptibility and further evaluate the likelihood of H5N1 HPAI viral maintenance in a wild bird population, we determined the concentration of virus required to produce infection in wood ducks. To accomplish this, 25 wood ducks were inoculated intranasally at 12-16 wk of age with decreasing concentrations of a H5N1 HPAI virus (A/Whooper Swan/Mongolia/244/05 [H5N1]). The median infectious dose and the lethal dose of H5N1 HPAI virus in wood ducks were very low (10(0.95) and 10(1.71) median embryo infectious dose [EID(50)]/ml, respectively) and less than that of chickens (10(2.80) and 10(2.80) EID(50)/ml). These results confirm that wood ducks are highly susceptible to infection with H5N1 HPAI virus. The data from this study, combined with what is known experimentally about H5N1 HPAI virus infection in wood ducks and viral persistence in aquatic environments, suggest that the wood duck would represent a sensitive indicator species for H5N1 HPAI. Results also suggest that the potential for decreased transmission efficiency associated with reduced viral shedding (especially from the cloaca) and a loss of environmental fitness (in water), may be offset by the ability of this virus to be transmitted through a very low infectious dose.     

Mosaic genome structure of simian immunodeficiency virus from west African green monkeys.

Elucidation of the phylogenetic origins of simian and human immunodeficiency viruses (SIV and HIV) is fundamental to the understanding of HIV pathogenesis and the spread of AIDS worldwide. In this study, we molecularly characterized multiple SIVAGM isolates from four different African green monkey species (vervet, grivet, sabaeus and tantalus monkeys). Phylogenetic analysis of partial (1 kb) env sequences indicated that all SIVAGM strains cluster together, and that they fall into four distinct sequence sub-groups according to their species of origin. However, alignment of long terminal repeat sequences revealed that SIVs from West African sabaeus monkeys contain a structural feature (a duplication of the transactivation response element) thus far only found in otherwise highly divergent lentiviruses infecting sooty mangabeys (SIVSM) and humans (HIV-2). To determine whether there were additional similarities with the SIVSM/HIV-2 group, a full-length replication competent sabaeus provirus was cloned and sequenced. In phylogenetic trees derived from the central and 3' coding regions, the sabaeus virus clustered with SIVAGM isolates from other African green monkey species. However, in trees derived from the 3' half of gag and the adjacent 5' region of pol, the sabaeus virus grouped with the SIVSM/HIV-2 lineage. These results indicated that the sabaeus virus comprised a mosaic genome which must have resulted from recombination of divergent lentiviruses in the distant past. A second, independent sabaeus isolate exhibited similar phylogenetic relationships, suggesting that all West African green monkey viruses share this complex evolutionary history. Taken together, these results indicate that African green monkeys have been infected with SIVAGM for very long periods of time, and that recombination and cross-species transmission in the wild have contributed to the genetic complexity of primate lentiviruses.     

Trichinella britovi etiological agent of sylvatic trichinellosis in the Republic of Guinea (West Africa) and a re-evaluation of geographical distribution for encapsulated species in Africa

In West Africa, Trichinella infection was documented in humans and animals from Senegal in the 1960s, and the biological characters of one isolate showed a lower infectivity to domestic pigs and rodents when compared with that of a Trichinella spiralis pig isolate from Europe. To identify the Trichinella species present in West Africa, a survey was conducted in a total of 160 wild animals in the Republic of Guinea. Three Viverridae, one true civet (Viverra civetta) and two African palm civets (Nandinia binotata) from the Fouta Djallon Massif, Pilimini Subprefecture, were found positive by artificial digestion of muscle samples. Trichinella larvae from these three viverrids were identified as Trichinella britovi and no difference was detected in three examined sequences from these African isolates and the reference strain of T. britovi from Europe, indicating common ancestry, an historically continuous geographic distribution, and recent isolation for African and European populations. The detection of T. britovi in West Africa modifies our knowledge about the distribution of encapsulated species of Trichinella in Africa. Thus, Trichinella nelsoni is now considered to have a distribution limited to the Eastern part of the Afrotropical region from Kenya to South Africa. This provides a plausible explanation for the presence of Trichinella T8 in Namibia and South Africa, and further suggests that T. britovi could be the Trichinella species circulating among wild animals of Northern Africa.     

Serological evidence of Ebola virus infection in Indonesian orangutans

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae and cause severe hemorrhagic fever in humans and nonhuman primates. Despite the discovery of EBOV (Reston virus) in nonhuman primates and domestic pigs in the Philippines and the serological evidence for its infection of humans and fruit bats, information on the reservoirs and potential amplifying hosts for filoviruses in Asia is lacking. In this study, serum samples collected from 353 healthy Bornean orangutans (Pongo pygmaeus) in Kalimantan Island, Indonesia, during the period from December 2005 to December 2006 were screened for filovirus-specific IgG antibodies using a highly sensitive enzyme-linked immunosorbent assay (ELISA) with recombinant viral surface glycoprotein (GP) antigens derived from multiple species of filoviruses (5 EBOV and 1 MARV species). Here we show that 18.4% (65/353) and 1.7% (6/353) of the samples were seropositive for EBOV and MARV, respectively, with little cross-reactivity among EBOV and MARV antigens. In these positive samples, IgG antibodies to viral internal proteins were also detected by immunoblotting. Interestingly, while the specificity for Reston virus, which has been recognized as an Asian filovirus, was the highest in only 1.4% (5/353) of the serum samples, the majority of EBOV-positive sera showed specificity to Zaire, Sudan, Cote d'Ivoire, or Bundibugyo viruses, all of which have been found so far only in Africa. These results suggest the existence of multiple species of filoviruses or unknown filovirus-related viruses in Indonesia, some of which are serologically similar to African EBOVs, and transmission of the viruses from yet unidentified reservoir hosts into the orangutan populations. Our findings point to the need for risk assessment and continued surveillance of filovirus infection of human and nonhuman primates, as well as wild and domestic animals, in Asia.     

The Ras-PI3K signaling pathway is involved in clathrin-independent endocytosis and the internalization of influenza viruses

Background

Influenza virus infection causes highly contagious, severe respiratory disorders and gives rise to thousands of deaths every year; however, the efficacy of currently approved defense strategies, including vaccines and neuraminidase inhibitors, is limited because the virus frequently acquires resistance via antigen drift and reassortment. It is therefore important to establish a novel, effective therapeutic strategy that is effective irrespective of viral subtype.

Methodology/Principal Findings

Here, we identify the Ras–phosphoinositide 3-kinase (PI3K) signaling pathway as a host-cell regulatory mechanism for influenza virus entry. The binding of Ras to PI3K is specifically involved in clathrin-independent endocytosis, endosomal maturation, and intracellular transport of viruses, which result in decreased infectious efficacy of different subtypes of influenza viruses in cells lacking the Ras–PI3K interaction. Moreover, influenza virus infection indeed triggered Ras activation and subsequent PI3K activation in early endosomes.

Conclusions/Significance

Taken together, these results demonstrate that the Ras–PI3K signaling axis acts as a host-oriented mechanism for viral internalization. Given that virus incorporation is a process conserved among virus subtypes and species, this signaling pathway may provide a target for potent, well-tolerated prophylactics and therapeutics against a broad range of viruses.