Isolation of a feline-derived feline panleukopenia virus with an A300P substitution in the VP2 protein and confrmation of its pathogenicity in dogs

Feline panleukopenia virus(FPV)is a single-stranded DNA virus that can infect cats and cause feline panleukopenia,which is a highly contagious and fatal disease in felines.The sequence of FPV is highly variable,and mutations in the amino acids of its capsid protein play crucial roles in altering viral virulence,immunogenicity,host selection,and other abilities.In this study,the epidemiology of FPV was studied using 746 gastrointestinal swab samples derived from cats that presented gastrointestinal symptoms specifcally,diarrhea or vomiting during the period spanning from 2018 to 2022.The overall prevalence of FPV-positive patients among these samples was determined to be 45.4%.Capsid(virion)protein 2(VP2)gene of each FPV-positive sample was sequenced and amplifed,yielding 65 VP2 sequences.Among them,six VP2 gene sequences were detected in the majority of the samples test positive for FPV,and these positive samples originated from a diverse range of geographical locations.These isolates were named FPV-6,FPV-10,FPV-15,FPV-251,FPV-271 and FPV-S2.Additionally,the substitution of Ala300Pro(A300P)in VP2 was detected for the frst time in feline-derived FPV(FPV-251).FPV-251 isolate,with this substitution in VP2 protein,exhibited stable proliferative capacity in Madin-Darby canine kidney(MDCK)cells and A72 cells.FPV-271 was selected as the FPV control isolate due to its single amino acid diference from VP2 protein of FPV-251 at position 300(FPV-271 has alanine,while FPV-251 has proline).After oral infection,both FPV-251 and FPV-271 isolates caused feline panleukopenia,which is characterized by clinical signs of enterocolitis.However,FPV-251 can infect dogs through the oral route and cause gastrointestinal(GI)symptoms with lesions in the intestine and mesenteric lymph nodes(MLNs)of infected dogs.This is the frst report on the presence of an A300P substitution in VP2 protein of feline-derived FPV.Additionally,FPV isolate with a substitution of A300P at VP2 protein demonstrated efcient replication capabilities in canine cell lines and the ability to infect dogs.

Endosomes and Microtubles are Required for Productive Infection in Aquareovirus

Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches.We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl,chloroquine and bafilomycin A1(Bafi).Confocal images indicated that GCRV particles could colocalize with Rab5,Rab7 and lysosomes in host cells.Further ultrastructural examination validated that viral particle was found in late endosomes.Moreover,disruption of microtubules with nocodazole clearly blocked GCRV entry,while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament.Notably,viral particles were observed to transport along microtubules by using QD-labeled GCRV.Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.

N-Terminal Myristoylated VP5 is Required for Penetrating Cell Membrane and Promoting Infectivity in Aquareoviruses

Dear Editor,Myristoylation is a naturally occurring post-translational modification for targeting cytoplasmic proteins to intracellular membranes.Unlike enveloped animal viruses,which enter host cells by membrane fusion,nonenveloped animal viruses must disrupt the cell membrane to initiate infection.Some animal viruses and several nonenveloped viruses

Identification and characterization of two cleavage fragments from the Aquareovirus nonstructural protein NS80

Aquareovirus species vary with respect to pathogenicity,and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly,which can form viral inclusion bodies(VIBs) and recruit viral proteins to its VIBs in infected cells.NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa.Interestingly,a short specific fragment of NS80 has also been detected in infected cells.In this study,an approximately58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80 C.Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose,suggesting that the fragment is not a transient intermediate of protein degradation.Moreover,another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80 N,indicating that the 58-kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80.Additionally,different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis,implying that the two cleavage fragments of NS80 function differently in the viral life cycle.These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

Functional Characterization of the Group Ⅰ Alphabaculovirus Specific Gene ac73

Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-,Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group Ⅰ and Ⅱ, and Group Ⅰ appears to be emerged most recently among all baculoviruses. Interestingly, there are 12 Group Ⅰ specific genes that are only found in this lineage. Studying these genes is helpful to understand how baculoviruses evolved. Here, we reported the functional analyzing results of ac73, a function unknown Group Ⅰ specific gene of Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) which is the type species of baculovirus. The AC73 protein encoded by ac73 was found to be expressed during the late stage of infection and incorporated into the nucleocapsids of budded virus(BV) and occlusionderived virus(ODV). In infected cells, AC73 resided mainly in the ring zone region of the nucleus, and appeared to be assembled into occlusion bodies(OBs). The ac73 knockout and repaired viruses were constructed and studied by in vitro and in vivo infection. Although ac73 was not essential for BV and ODV or OB formation, the BV titer and viral infectivity in insect larvae of ac73 knockout Ac MNPV decreased by about 5–8 and 3–4 fold compared to those of wild type virus,respectively, suggesting ac73 contributed to infectious BV production and viral infectivity in vivo. This research provides new insight into the function of this Group I specific gene.

Characterization of viral entry and infection of quantum dot-labeled grass carp reovirus

Dear Editor,Reoviruses are non-enveloped icosahedral virions with an outer capsid surrounding their cores,which harbor the10–12 segmented double-stranded RNA(ds RNA)genome.To date,there are 15 proposed genera in the family Reoviridae(King et al.,2012),including Aquareovirus.Generally,aquareoviruses are of low pathogenicity in aquaculture.However,grass carp reovirus(GCRV)is