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.     

Further Studies on the Replication of Rabies and Rabies-Like Viruses in Organized Cultures of Mammalian Neural Tissues

Organized cultures of mammalian spinal and dorsal root ganglions were used for a comparative study of the neurocytopathology caused by rabies and so-called rabies-like viruses. Electron microscopy and titration of infectivity revised earlier data in which no difference could be demonstrated between street and fixed-virus infection. In the present study, fixed virus produced inclusion bodies without apparent virus assembly. Sequential electron microscopy revealed that the main sites of virus assembly were the membranes of the Golgi complex. In contrast, rabies-like viruses freshly isolated from wild rodents produced inclusion bodies all of which were associated with virus replication. Electron microscopic evidence has led us to classify these strains as street virus. Nonneural cell elements from cultivated ganglions were susceptible to fixed virus and the cultures yielded higher titers of infectivity as compared to those of rabies-like viruses. Virus budding was shown to occur at the cell surface as well as at intracytoplasmic membranes.     

Viral replication, persistence in water and genetic characterization of two influenza A viruses isolated from surface lake water

Water-borne transmission has been suggested as an important transmission mechanism for Influenza A (IA) viruses in wild duck populations; however, relatively few studies have attempted to detect IA viruses from aquatic habitats. Water-isolated viruses have rarely been genetically characterized and evaluation for persistence in water and infectivity in natural hosts has never been documented. In this study, we focused on two IA viruses (H3N8 and H4N6 subtypes) isolated from surface lake water in Minnesota, USA. We investigated the relative prevalence of the two virus subtypes in wild duck populations at the sampling site and their genetic relatedness to IA viruses isolated in wild waterbirds in North America. Viral persistence under different laboratory conditions (temperature and pH) and replication in experimentally infected Mallards (Anas platyrhynchos) were also characterized. Both viruses were the most prevalent subtype one year following their isolation in lake water. The viruses persisted in water for an extended time period at constant temperature (several weeks) but infectivity rapidly reduced under multiple freeze-thaw cycles. Furthermore, the two isolates efficiently replicated in Mallards. The complete genome characterization supported that these isolates originated from genetic reassortments with other IA viruses circulating in wild duck populations during the year of sampling. Based on phylogenetic analyses, we couldn't identify genetically similar viruses in duck populations in the years following their isolation from lake water. Our study supports the role for water-borne transmission for IA viruses but also highlights that additional field and experimental studies are required to support inter-annual persistence in aquatic habitats.     

Reassortant influenza A viruses in wild duck populations: effects on viral shedding and persistence in water

Wild ducks of the genus Anas represent the natural hosts for a large genetic diversity of influenza A viruses. In these hosts, co-infections with different virus genotypes are frequent and result in high rates of genetic reassortment. Recent genomic data have provided information regarding the pattern and frequency of these reassortant viruses in duck populations; however, potential consequences on viral shedding and maintenance in the environment have not been investigated. On the basis of full-genome sequencing, we identified five virus genotypes, in a wild duck population in northwestern Minnesota (USA), that naturally arose from genetic reassortments. We investigated the effects of influenza A virus genotype on the viral shedding pattern in Mallards (Anas platyrhynchos) and the duration of infectivity in water, under different temperature regimens. Overall, we found that variation in the viral genome composition of these isolates had limited effects on duration, extent and pattern of viral shedding, as well as on the reduction of infectivity in water over time. These results support that, in wild ducks, functionally equivalent gene segments could be maintained in virus populations with no fitness costs when genetic reassortments occur.     

Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme

The use of baculoviruses as biological control agents is hampered by their susceptibility to inactivation by ultraviolet (UV) light. In an attempt to reduce UV inactivation, an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, was expressed in the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV), and the infectivity of recombinant viruses expressing cv-PDG was measured after exposure to UV light. Expression of cv-PDG resulted in a 3-fold decrease in inactivation of budded virus by UV as measured by plaque assay in Spodoptera frugiperda Sf21 cells. However, occluded viruses expressing cv-PDG were not more resistant to UV inactivation than wild type AcMNPV when fed to either S. frugiperda or Trichoplusia ni neonate larvae. Surprisingly, however, viruses expressing cv-PDG showed a significant decrease in both the dose of occluded virus required for oral lethality and the time required for lethality compared to control virus, but these effects were only seen in S. frugiperda and not in T. ni larvae.     

Filter-feeding bivalves can remove avian influenza viruses from water and reduce infectivity

Avian influenza (AI) viruses are believed to be transmitted within wild aquatic bird populations through an indirect faecal–oral route involving contaminated water. This study examined the influence of filter-feeding bivalves, Corbicula fluminea, on the infectivity of AI virus in water. Clams were placed into individual flasks with distilled water inoculated 1:100 with a low pathogenic (LP) AI virus (A/Mallard/MN/190/99 (H3N8)). Viral titres in water with clams were significantly lower at 24 and 48 h post-inoculation compared to LPAI-infected water without clams. To determine whether clams affected the infectivity of AI viruses, 18 wood ducks (Aix sponsa) were divided into test groups and inoculated with a variety of treatments of clam supernatants, whole clams and water exposed to a high pathogenic (HP) AI (A/whooper swan/Mongolia/244/05 (H5N1)). None of the wood ducks inoculated with HPAI-infected water that was filtered by clams or that was inoculated with or fed tissue from these clams exhibited morbidity or mortality. All wood ducks exposed to either HPAI-infected water without clams or the original viral inoculum died. These results indicate that filter-feeding bivalves can remove and reduce the infectivity of AI viruses in water and demonstrate the need to examine biotic environmental factors that can influence AI virus transmission.     

Role for influenza virus envelope cholesterol in virus entry and infection

Enveloped viruses are highly dependent on their lipid envelopes for entry into and infection of host cells. Here, we have examined the role of cholesterol in the virus envelope, using methyl-beta-cyclodextrin depletion. Pretreatment of virions with methyl-beta-cyclodextrin efficiently depleted envelope cholesterol from influenza virus and significantly reduced virus infectivity in a dose-dependent manner. A nonenveloped virus, simian virus 40, was not affected by methyl-beta-cyclodextrin treatment. In the case of influenza virus, infectivity could be partially rescued by the addition of exogenous cholesterol. Influenza virus morphology, binding, and internalization were not affected by methyl-beta-cyclodextrin depletion, whereas envelope cholesterol depletion markedly affected influenza virus fusion, as measured by a specific reduction in the infectivity of viruses induced to fuse at the cell surface and by fluorescence-dequenching assays. These data suggest that envelope cholesterol is a critical factor in the fusion process of influenza virus.     

Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.     

Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

Background

Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus.

Methodology and Principal findings

We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses.

Conclusions

These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.     

Insertion of a classical nuclear import signal into the matrix domain of the Rous sarcoma virus Gag protein interferes with virus replication

The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a "classical " nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.     

The deletion of the pif gene improves the biosafety of the baculovirus-based technologies

Our goal was to improve the biosafety of baculovirus-based technologies by deleting the pif (per os infectivity factor) gene from baculovirus expression vectors. Such a deletion would block transmission in nature without disturbing protein production. A pif deletion mutant of Autographa californica multiplecapsid nucleopolyhedrovirus (AcMNPV) was constructed and its infectivity to two host species was tested by oral or intrahemocoelic inoculation. Virus replication after oral inoculation was monitored using PCR. Oral inoculations with a mixture of the wild type and the pif deletion viruses were carried out. The pif deletion blocked oral infection but it did not hamper infectivity in cell culture. The blockage took place early after inoculation and could not be overcome by mixed inoculations with the wild type. The cat gene was inserted under the control of the polyhedrin promoter in the deletion mutant and the wild type CAT yield was measured in Spodoptera frugiperda insect cells (Sf9) infected with either recombinant. The pif deletion did not hamper CAT production. This deletion significantly improved CAT yields early in the infection. Hence, expression vectors lacking pif may produce higher quality protein. The pif deletion is a simple measure that dramatically reduces the chances of virus spread or gene transfer in nature.     

Persistence of avian influenza viruses in lake sediment, duck feces, and duck meat

The persistence of 3 low-pathogenicity avian influenza viruses (LPAIV) (H4N6, H5N1, and H6N8) and one human influenza virus (H1N1) as well as Newcastle disease virus (NDV) and enteric cytopathogenic bovine orphan (ECBO) virus was investigated in lake sediment, duck feces, and duck meat at 30, 20, 10, and 0°C using a germ carrier technique. Virus-loaded germ carriers were incubated in each substrate, and residual infectivity of the eluted virus was quantified on cell culture after regular intervals for a maximum of 24 weeks. Data were analyzed by a linear regression model to calculate T(90) values (time required for 90% loss of virus infectivity) and estimated persistence of the viruses. In general, the persistence of all of the viruses was highest in lake sediment, followed by feces, and was the lowest in duck meat at all temperatures. For the avian influenza virus subtypes, T(90) values in sediment ranged from 5 to 11, 13 to 18, 43 to 54, and 66 to 394 days at 30, 20, 10, and 0°C, respectively, which were 2 to 5 times higher than the T(90) values of the viruses in the feces and meat. Although the individual viruses vary in tenacity, the survival time of influenza viruses was shorter than that of NDV and ECBO virus in all substrates. The results of this study suggest that lake sediment may act as a long-term source of influenza viruses in the aquatic habitat, while the viruses may remain infectious for extended periods of time in duck feces and meat at low temperatures, allowing persistence of the viruses in the environment over winter.     

Electron Microscopy of a Plant-Pathogenic Virus in the Nervous System of Its Insect Vector

The central nervous system of wound tumor virus (WTV)-infected Agallia constricta was studied by electron microscopy to obtain information concerning the virus distribution in the nervous system. Wound tumor virions were mostly found in the cytoplasm of the ganglion cells and less frequently in the glial cells. WTV was occasionally observed in the perineurium cells, nerve axons, tracheoblasts, and lateral nerves. In the ganglion cells, virions appeared as individual isolated particles (V(1)), in tubular formation (V(2)), and occasionally in aggregates (V(3)). In the glial cells, the virions were mostly seen in the V(3) formation, and very seldom in the V(1) and V(2) formations. In the perineurium cells and tracheoblasts, only small V(3) formations were observed. The isolated virions were usually surrounded with polyribosomes, and often appeared around the foci of the viroplasm. Sometimes degenerating ganglion cells infected with the WTV were encountered. These damaged cells strongly indicated that WTV exerted a cytopathogenic effect on the nerve cells.     

In vitro studies of the mechanism of leukemogenesis. II. Characterization of endogenous murine leukemia viruses isolated from AKR thymic epithelial reticulum cell lines

Thymic epithelial reticulum (TER) cell lines were established from thymuses of a young healthy AKR mouse (A2T), a preleukemic AKR mouse (A6T), and two lymphoma-bearing AKR/Ms mice (ASLT-1 and ASLT-2). Numerous type-C virus particles with occasional budding forms were observed in all cell lines. Expression of XC-detectable, N-tropic, ecotropic virus was observed in every cell line, whereas the presence of xenotropic and mink cell focus-inducing (MCF) viruses could be detected only in TER cells derived from preleukemic and leukemic mice. Expression of xenotropic virus in various cells of newborn and young AKR mice could readily be induced by IUdR treatment, whereas MCF virus was never detected in these cells, with the exception of the A2T cell line after more than 20 passages, in which MCF virus with dual-tropic infectivity emerged in addition to ecotropic and xenotropic viruses. These spontaneous and induced MCF viruses were purified, and their virological properties were characterized. The cloned MCF viruses (MCFs AT1, AT2, AT3, and AT4-IU) showed dual tropism and produced cytopathic effect-like foci in mink lung cells. Preinfection with either ecotropic or xenotropic virus interfered with the infectivity of MCF viruses. Spontaneous leukemogenesis in AKR mice was accelerated by the inoculation of MCF viruses. These findings indicate that TER cells could serve as the host cells for the genetic recombination of the endogenous MuLV; the recombinant MuLV, MCF virus, appears to be most closely associated with leukemogenesis in AKR mice.     

Tmprss2 Is Essential for Influenza H1N1 Virus Pathogenesis in Mice

Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.     

The C terminus of human immunodeficiency virus type 1 matrix protein is involved in early steps of the virus life cycle.

Deletion mutations at the C terminus of the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) were generated by site-directed mutagenesis. The resultant mutant viruses had a severe defect in virus infectivity. This defect did not involve late steps of the virus life cycle, as the synthesis and processing of the Gag polyprotein and the assembly and release of mutant virions were not greatly affected. The incorporation of viral proteins and the viral RNA genome was similar for mutant and wild-type virions. In contrast, the early steps of the virus life cycle were severely affected, as the synthesis of viral DNA postinfection was dramatically reduced in mutant-virus-infected cells. One stretch of amino acids that was deleted in one of the mutants has significant homology with a region in VP1 of the picornavirus family. This region of VP1 is presumably involved in poliovirus penetration into cells. These results suggest that in addition to its functional role in virus assembly, the MA protein of HIV-1, and possibly of other retroviruses, plays an important role in virus entry.     

Role of human immunodeficiency virus type 1 matrix phosphorylation in an early postentry step of virus replication

The matrix domain (MA) is important for targeting of human immunodeficiency virus type 1 Gag assembly to the plasma membrane, envelope incorporation into virions, preintegration complex import into the nucleus, and nuclear export of viral RNA. Myristylation and phosphorylation are key regulatory events for MA function. Previous studies have indicated that MA phosphorylation at serine (Ser) residues is important for viral replication. This study defines the molecular mechanisms of virus particle assembly and infectivity through a detailed study of the role of MA serine phosphorylation. We show that the combined mutation of Ser residues at positions 9, 67, 72, and 77 impairs viral infectivity in dividing and nondividing cells, although the assembly of these Ser mutant viruses is comparable to that of wild-type virus. This defect can be rescued by pseudotyping these mutant viruses with vesicular stomatitis virus G protein, suggesting that these serine residues are critical in an early postentry step of viral infection. The phosphorylation level of MA in defective mutant viruses was severely reduced compared to that of the wild type, suggesting that phosphorylation of Ser-9, -67, -72, and -77 is important for an early postentry step during virus infection.     

SOME EFFECTS OF TANNIC ACID AND OF LEAF EXTRACTS WHICH CONTAIN TANNINS ON THE INFECTIVITY OF TOBACCO MOSAIC AND TOBACCO NECROSIS VIRUSES

The inhibition of infection by tobacco necrosis and tobacco mosaic viruses by tannic acid, and by extracts of raspberry and strawberry leaves, was associated with the precipitation of the viruses. Precipitation and inhibition were reversible, and infective virus was obtained from the precipitate formed between the viruses and tannins. Infectivity was fully restored by diluting mixtures of virus and tannin adequately and partially restored by adding alumina or nicotine sulphate.
Viruses and tannins are thought to form non-infective complexes, in which the virus and tannin components are held together by co-ordinate linkages or hydrogen bonds.
Macerating tobacco leaves infected with tobacco mosaic virus together with raspberry leaves greatly decreased the infectivity of the extracts; adding nicotine sulphate to the mixture of leaves before it was ground increased the infectivity, even though nicotine sulphate alone decreases the infectivity of tobacco mosaic virus. Even in the presence of nicotine sulphate, much of the virus was precipitated by substances from the raspberry leaves.
Extracts of roots of Fragaria vesca plants, infected with a tobacco necrosis virus, were more infective when made by macerating the roots with four times their weight of buffer at pH 8 than when made without buffer. Various methods are suggested for facilitating the transmission of viruses from plants that contain tannin.     

Genome composition analysis of the reassortant influenza viruses used in seasonal and pandemic live attenuated influenza vaccine

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.