The Conserved Herpesviridae Protein Kinase (CHPK) of Gallid alphaherpesvirus 3 (GaHV3) Is Required for Horizontal Spread and Natural Infection in Chickens

Abstract

We have formerly identified the conserved herpesvirus protein kinase (CHPK) as essential for horizontal transmission of Marek’s disease virus (MDV). Thus far, it has been confirmed that the mutation of the invariant lysine (K) of CHPKs abrogates kinase activity and that CHPK activity is required for MDV horizontal transmission. Since CHPK is conserved among all members of the Herpesviridae, we hypothesized that CHPK, and specifically its kinase activity, is important for the horizontal transmission of other herpesviruses. To test this hypothesis, we utilized our experimental and natural infection model in chickens with MD vaccine strain 301B/1 of Gallid alphaherpesvirus 3 (GaHV3). First, we mutated the invariant lysine (K) 157 of 301B/1 CHPK to alanine (A) and determined whether it was required for horizontal transmission. To confirm the requirement of 301B/1 CHPK activity for transmission, a rescued virus was generated in which the A157 was changed back to a K (A157K). Despite both the CHPK mutant (K157A) and rescuant (A157K) viruses having replication defects in vivo, only the CHPK mutant (K157A) was unable to spread to contact chickens, while both wild-type and rescuant (A157K) viruses transmitted efficiently, confirming the importance of CHPK activity for horizontal spread. The data confirm that CHPK is required for GaHV3 transmission and suggest that the requirement of avian CHPKs for natural infection is conserved.

Coinfection in the host can result in functional complementation between live vaccines and virulent virus

“…A summary of viruses used in this study are shown along with plaques formed during coinfection in cell culture showing both viruses present in the mixture ( Figure 2 ). Chickens were inoculated with a mixture of MDV-NT (lacking MDV gC) and vacMD-T (vacMD expressing MDV gC) or vacMD-NT (vacMD that has CHPK mutated) of which all viruses have previously been published for their transmissibility [ 17 , 18 , 20 ]. MDV-NT lacks gC, required for transmission and expresses enhances green fluorescent protein (eGFP), while both vacMD-T and vacMD-NT express monomeric red fluorescent protein (mRFP) allowing us to differentiate each virus in cell culture and in vivo .…”

“…MDV-NT lacks gC (MDV δgc) that is required for transmission [ 8 , 9 , 23 ]. vacMD was rendered NT through mutation of its CHPK activity by mutation of the invariant lysine (K157A) that is required for transmission [ 18 ]. vacMD-T expresses MDV gC in place of its gC [ 17 ].…”

“…Three-day old specific pathogen free (SPF) Pure Columbian (PC) chickens were obtained from the UIUC Poultry Farm (Urbana, IL) and food and water were provided ad libitum for all bird experiments. Viruses used in this study have been previously described [ 17 , 18 , 20 ]. Previously titrated cell-associated MDV and vacMD were diluted and mixed prior to injection into chickens by intra-abdominal inoculation to establish experimental coinfection (Experiment 1, n = 12; Experiment 2, n = 13).…”

“…Following inoculation, viruses were back tittered and were as follows: For Experiment 1, 2,000 and 7,000 plaque forming units (PFU) of non-transmissible MDV (MDV-NT) and transmissible MD vaccine (vacMD-T), respectively, were inoculated into chicks, while in Experiment 2, 3,000 and 6,000 PFU of MDV-NT and non-transmissible MD vaccine (vacMD-NT), respectively, were used. The higher dose of vacMD used was based on previous studies in which this vaccine requires higher doses that MDV for establishing infection [ 18 , 21 ].…”

“…Importantly, MD vaccines are currently being used as vaccine vectors to deliver antigens of other pathogens, such as avian influenza (AIV), infectious laryngotracheitis (ILTV), Newcastle disease (NDV), or infectious bursal disease (IBDV) viruses [ 16 ]. We have recently shown that MD vaccine GaHV3 strain 301B/1 has a similar viral life cycle as MDV in which gC [ 17 ] and CHPK [ 18 ] are required for natural infection.…”