Development and pathogenicity analysis of full-length infectious cDNA clones of citrus yellow mottle-associated virus in citrus plants

Citrus yellow mottle-associated virus(CiYMaV)belonging to the subgenus Mandarivirus within the genus Potexvirus,was first identified in 2018 from Pakistan(CiYMaV-PK),where it is endemic in several regions.Here,three full-length cDNA clones(pCiYMaV-FL-1,pCiYMaV-FL-18,and pCiYMaV-FL-22)corresponding to the genomic RNA of CiYMaV were constructed and then agroinfiltrated on Chandler pummelo(Citrus grandis)seedlings using the vacuum infiltration method.All the inoculated plants developed severe vein yellowing,leaf mottling,and dwarfing symptoms by 40 days post-infiltration(dpi).The results of a direct tissue blot immunoassay and reverse transcription polymerase chain reaction detection showed 94.7–100%infection rates of pCiYMaV-FL at 60 dpi.Despite there being no observed difference among the three clones in the severity of symptom,pCiYMaV-FL-22 showed the highest accumulation levels of viral RNA and coat proteins.Moreover,pCiYMaV-FL-22 successfully infected seven other citrus varieties and induced symptoms in five of them.Transmission electron microscopy identified the presence of filamentous virus particles in extracts from systemic leaves of the plants infected with pCiYMaV-FL-22 at 6-months post-infiltration.Taken together,the results indicate that Koch's postulates were fulfilled for CiYMaV in citrus plants.This is the first report of full-length infectious cDNA clones of CiYMaV,and thus,the data provide a basis for further study of the molecular mechanisms of virus infection and the development of a viral vector to express foreign genes in citrus plants.

MORPHOLOGICAL SURVEY ON ENDOGENOUS C-TYPE VIRUSES INFECTING A HUMAN LUNG SQUAMOUS CARCINOMA PASSAGED IN NUDE MICE

A human lung squamous carcinoma was transplanted and passaged in Swiss-DF nude mice, called LSX-83, for more than five years in our laboratory. The morphological characteristics of the original tumor were maintained in passages from 4 to 33. But from the 35th generation, an increasing amount of tonofilaments and nuclear segregation with typical features was found with electron microscopy. The C-type virus particles were first detected in extra cellular space after 40 passages. The viruses were observed in different stages of growth, but their distribution and number did not show apparent change up to 54 passages. Such findings suggest that LSX-83 cells probably possess certain barrier of resistance against C-type viruses. The relation between C-type viruses and the morphological changes of LSX-83 cells was discussed.

MORPHOLOGY AND MORPHOGENESIS OF HEPATITIS E VIRUS (STRAIN 87A)

The morphology and morphogenesis of isolated hepatitis E virus (HEV, strain 87A) were observed by electron microscopy (EM) and immune electron microscopy (IEM). Progressively developing local vesicles, virions accumulation in crystalline arrays and viroplasmic focus were seen in cytoplasm of infected cells. Replication and assembly of the new generation viruses were closely associated with rough endoplasmic reticulum (RER), inclusion body (IB) and microfibrils. Condensation and margination of chromatin, dispersion of nucleolar material, nuclear membrane alteration and masses of threads, granular material, and fibrillar component of the nucleus were frequently found. These changes revealed that this strain virus was confirmed as a RNA virus. The shape of the virus particles appeared approximately spherical whether the specimens were from the tissue culture crude suspension or purified highly concentrated preparations. The size of the virion was about 30 nm in diameter. The viral particles appeared unsmooth and irregular in outline. The spike-like structures may be occationally observed on the surface of some viral capsides. The diameter of the strain 87 A virus is larger than the picornavirus and smaller than the calicivirus. This strain virus is different from classical calicivirus in without the cup-shaped surface depressions. The new genus, heparnavirus genus of caliciviridae family should be proposed for HEV.