Yongkun Zhao

Domestic cats and dogs are susceptible to H9N2 avian influenza virus

Replication and transmission of avian influenza virus (AIV) in domestic dogs and cats may pose a risk to humans. The susceptibility of cats and dogs to H9N2 influenza virus was evaluated by intranasally or orally inoculating animals with an H9N2 influenza virus. Cats had recoverable virus in respiratory tissues and the olfactory bulb three days post-inoculation and shed H9N2 virus into nasal washes and pharyngeal swabs from day 2 through day 10 post-inoculation. Virus was recovered from respiratory tissues of dogs three days post-inoculation, but was not detected in nasal washes or pharyngeal swabs. While no virus shedding or replication was detected in cats or dogs following consumption of H9N2-infected chicks, one of two cats and one of two dogs seroconverted. Two of three naïve contact cats seroconverted following co-housing with cats that were intranasally inoculated with H9N2 virus, whereas none of the three naïve contact dogs seroconverted. Our results demonstrate that H9N2 AIV can infect domestic cats and dogs via the upper respiratory tract and indicate that cats are more susceptible than dogs to H9N2 AIV. These findings suggest that domestic dogs and cats may serve as host species contributing to the adaptation of H9N2 viruses in mammals.

Experimental infection of non-human primates with avian influenza virus (H9N2).

Several cases of humans infected with the H9N2 avian influenza virus (AIV) have been described since 1999; however, the infectivity and pathogenicity of H9N2 in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2 virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2 AIV presented with biphasic fever and viral pneumonia. H9N2 was recovered from nasal washes and pharyngeal samples up to days 7-9 postinfection, followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2 AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2 influenza virus infections, particularly in the context of viral evolution and pathogenicity.

MERS-CoV virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans with a case fatality rate of over 39%, and poses a considerable threat to public health. A lack of approved vaccine or drugs currently constitutes a roadblock in controlling disease outbreak and spread. In this study, we generated MERS-CoV VLPs using the baculovirus expression system. Electron microscopy and immunoelectron microscopy results demonstrate that MERS-CoV VLPs are structurally similar to the native virus. Rhesus macaques inoculated with MERS-CoV VLPs and Alum adjuvant induced virus-neutralizing antibodies titers up to 1:40 and induced specific IgG antibodies against the receptor binding domain (RBD), with endpoint titers reaching 1:1,280. MERS-CoV VLPs also elicited T-helper 1 cell (Th1)-mediated immunity, as measured by ELISpot. These data demonstrate that MERS-CoV VLPs have excellent immunogenicity in rhesus macaques, and represent a promising vaccine candidate.

Treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from Ebola virus infection

Recent successes with monoclonal antibody cocktails ZMappTM and MIL77 against Ebola virus (EBOV) infections have reignited interest in antibody-based therapeutics. Since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. We produced purified equine antisera from horses hyperimmunized with EBOV virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. BALB/c mice were given up to 2 mg of purified equine antisera per animal, at 30 minutes, 1 or 2 days post-infection (dpi), in which all animals survived. To decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate F(ab′)2 fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. Full protection was achieved with when treatment was initiated at 1 dpi, but the suboptimal protection observed with the 30 minute and 2 dpi groups demonstrate that in addition to virus neutralization, other Fc-dependent antibody mechanisms may also contribute to survival. Guinea pigs given 20 mg of antisera or F(ab′)2 at or starting at 1 or 2 dpi were also fully protected from EBOV infection. These results justify future efficacy studies for purified equine products in NHPs.

Adaptive amino acid substitutions enhance the virulence of a reassortant H7N1 avian influenza virus isolated from wild waterfowl in mice

H7 avian influenza viruses (AIVs) have caused a number of human infections, highlighting the pandemic potential of them. However, the factors that promote their replication in mammals remain poorly understood. Here, we generated mouse-adapted variants of a reassortant H7N1 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the variants were reduced >10,000-fold compared to the parental virus. Adapted variants displayed enhanced replication kinetics in vitro and vivo, and were capable of replicating in multiple organs. Analysis of the variant virus genomes revealed amino acid changes in the PB2 (E627K), HA (H3 numbering; E114K, G205E, and G218E), and NA (S350N) proteins. Notably, some amino acid changes have been identified in natural H7 isolates. Our results implicate a number of amino acid substitutions that collectively enhance the ability of a wild bird-origin H7N1 AIV to replicate and cause severe disease in mice.

Adaptive amino acid substitutions enhance the virulence of an H7N7 avian influenza virus isolated from wild waterfowl in mice

Although H7N7 AIVs primarily circulate in wild waterfowl, documented cases of human infection with H7N7 viruses suggest they may pose a pandemic threat. Here, we generated mouse-adapted variants of a wild waterfowl-origin H7N7 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the adapted variants were reduced >5000-fold compared to the parental virus. Mouse-adapted variants viruses displayed enhanced replication in vitro and in vivo, and acquired the ability to replicate in extrapulmonary tissues. These observations suggest that enhanced growth characteristics and modified cell tropism may increase the virulence of H7N7 AIVs in mice. Genomic analysis of the adapted variant viruses revealed amino acid changes in the PB2 (E627K), PB1 (R118I), PA (L550M), HA (G214R), and NA (S372N) proteins. Our results suggest that these amino acid substitutions collaboratively enhance the ability of H7N7 virus to replicate and cause severe disease in mammals.

Adaptation of H9N2 AIV in guinea pigs enables efficient transmission by direct contact and inefficient transmission by respiratory droplets.

H9N2 avian influenza viruses circulate worldwide in poultry and have sporadically infected humans, raising concern whether H9N2 viruses have pandemic potential. Here, we use a guinea pig model to examine whether serial passage results in adaptive viral changes that confer a transmissible phenotype to a wild-type H9N2 virus. After nine serial passages of an H9N2 virus through guinea pigs, productive transmission by direct contact occurred in 2/3 guinea pig pairs. The efficiency of transmission by direct contact increased following the fifteenth passage and occurred in 3/3 guinea pig pairs. In contrast, airborne transmission of the passaged virus was less efficient and occurred in 1/6 guinea pig pairs and 0/6 ferret pairs after the fifteenth passage. Three amino acid substitutions, HA1-Q227P, HA2-D46E, and NP-E434K, were sufficient for contact transmission in guinea pigs (2/3 pairs). The two HA amino acid substitutions enhanced receptor binding to α2,3-linked sialic acid receptors. Additionally, the HA2-D46E substitution increased virus thermostability whereas the NP-E434K mutation enhanced viral RNA polymerase activity in vitro. Our findings suggest that adaptive changes that enhance viral receptor binding, thermostability, and replicative capacity in mammalian cells can collectively enhance the transmissibility of H9N2 AIVs by direct contact in the guinea pig model.

Identification and Characterization of Porcine Kobuvirus Variant Isolated from Suckling Piglet in Gansu Province, China

Kobuviruses comprise three species, the Aichivirus A, Aichivirus B, and Aichivirus C (porcine kobuvirus). Porcine kobuvirus is endemic to pig farms and is not restricted geographically but, rather, is distributed worldwide. The complete genomic sequences of four porcine kobuvirus strains isolated during a diarrhea outbreak in piglets in the Gansu province of China were determined. Two of these strains exhibited variations relative to the traditional strains. The potential 3C/3D cleavage sites of the variant strains were Q/C, which differed from the Q/S in the traditional porcine kobuvirus genome. A 90-nucleotide deletion in the 2B protein and a single nucleotide insertion in the 3′UTR were found in the variant strains. The VP1 regions of all four porcine kobuviruses in our study were highly variable (81%–86%). Ten common amino acid mutations were found specifically at certain positions within the VP1 region. Significant recombination sites were identified using SimPlot scans of whole genome sequences. Porcine kobuviruses were also detected in pig serum, indicating that the virus can escape the gastrointestinal tract and travel to the circulatory system. These findings suggest that mutations and recombination events may have contributed to the high level of genetic diversity of porcine kobuviruses and serve as a driving force in its evolution.

H5N1 influenza virus-like particle vaccine protects mice from heterologous virus challenge better than whole inactivated virus

The highly pathogenic avian influenza (HPAI) H5N1 virus has become highly enzootic since 2003 and has dynamically evolved to undergo substantial evolution. Clades 2.3.2.1 and 2.3.4 have become the most dominant lineage in recent years, and H5N8 avian influenza outbreaks have been reported Asia. The current approach to generate influenza virus vaccines uses embryonated chicken eggs for large-scale production, although such vaccines have been poorly immunogenic to heterologous virus challenge. In the current study, virus-like particles (VLP) based on A/meerkat/Shanghai/SH-1/2012 (clade 2.3.2.1) and comprising hemagglutinin (HA), neuraminidase (NA), and matrix (M1) were produced using a baculovirus expression system to develop effective protection for different H5 HPAI clade challenges. Mice immunized with VLP demonstrated stronger humoral and cellular immune responses than mice immunized with whole influenza virus (WIV), with 20-fold higher IgG antibody titers against A/meerkat/Shanghai/SH-1/2012 after boost. Notably, the WIV vaccine group showed partial protection (80% survival) to homologous challenge, little protection (40% survival) to heterologous challenge, and 20% survival to H5N8 challenge, whereas all mice in the VLP+CFA group survived. These results provide insight for the development of effective prophylactic vaccines based on VLPs with cross-clade protection for the control of current H5 HPAI outbreaks in humans.

Canine Parvovirus VP2 Protein Expressed in Silkworm Pupae Self-Assembles into Virus-Like Particles with High Immunogenicity

The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4+ and CD8+ T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.